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Chronic inflammation of adipose tissue is a prominent characteristic of many metabolic diseases. Lipid metabolism in adipose tissue is consistently dysregulated during inflammation, which is characterized by substantial infiltration by proinflammatory cells and high cytokine concentrations. Adipose tissue inflammation is caused by a variety of endogenous factors, such as mitochondrial dysfunction, reactive oxygen species (ROS) production, endoplasmic reticulum (ER) stress, cellular senescence, ceramides biosynthesis and mediators of lipopolysaccharides (LPS) signaling. Additionally, the gut microbiota also plays a crucial role in regulating adipose tissue inflammation. Essentially, adipose tissue inflammation arises from an imbalance in adipocyte metabolism and the regulation of immune cells. Specific inflammatory signals, including nuclear factor-κB (NF-κB) signaling, inflammasome signaling and inflammation-mediated autophagy, have been shown to be involved in the metabolic regulation. The pathogenesis of metabolic diseases characterized by chronic inflammation (obesity, insulin resistance, atherosclerosis and nonalcoholic fatty liver disease [NAFLD]) and recent research regarding potential therapeutic targets for these conditions are also discussed in this review., (© 2024 World Obesity Federation.)

Fat grafting is a promising technique for correcting soft tissue abnormalities, but oil cyst formation and graft fibrosis frequently impede the therapeutic benefit of fat grafting. The lipolysis of released oil droplets after grafting may make the inflammation and fibrosis in the grafts worse; therefore, by regulating adipose triglyceride lipase (ATGL) via Atglistatin (ATG) and Forskolin (FSK), we investigated the impact of lipolysis on fat grafts in this study. After being removed from the mice and chopped into small pieces, the subcutaneous fat from wild-type C57BL/6J mice was placed in three different solutions for two hours: serum-free cell culture medium, culture medium+FSK (50 μM), and culture medium+ATG (100 μM). Following centrifugation to remove water and free oil droplets, 0.3 mL of the fat particles per mouse was subcutaneously injected into the back of mice. Additionally, the subcutaneous fat grafting area was immediately injected with PBS (control group), ATG (30 mg/kg), and FSK (15 mg/kg) following fat transplantation. Detailed cellular events after grafting were investigated by histological staining, real-time polymerase chain reaction, immunohistochemistry/immunofluorescent staining, and quantification. Two weeks after grafting, grafts treated with ATG showed lower expression of ATGL and decreased mRNA levels of TNFα and IL-6. In contrast, grafts treated with ATG showed elevated expression levels of IL-4 and IL-13 compared to the control grafts. In addition, fewer apoptotic cells and oil cysts were observed in ATG grafts. Meanwhile, a higher CD206+/CD68+ ratio of macrophages and more CD31+ vascular endothelial cells existed in the 2-month ATG grafts. In comparison to the control, ATG treatment improved the volume retention of grafts, and decreased graft fibrosis and oil cyst formation. By preventing oil droplet lipolysis, pharmacological suppression of ATGL shielded adipocytes from lipotoxicity following grafting. Additionally, ATG ameliorated the apoptosis and inflammation brought on by adipocyte death and oil droplet lipolysis in grafted fat. These all indicate that lipolysis inhibition improved transplanted fat survival and decreased the development of oil cysts and graft fibrosis, offering a potential postoperative pharmacological intervention for bettering fat grafting., (© 2024 Federation of American Societies for Experimental Biology.)

In this report, we present a case study of a rare human bacterium, Corynebacterium bovis , which caused an infection in a patient who had undergone autologous fat-based breast augmentation using cryopreserved fat. This infection occurred during a secondary fat grafting procedure. To identify the bacteria causing the infection, we used high-throughput DNA sequencing technology since this bacterium is seldomly reported in human infections. The patient was successfully treated with intravenous imipenem. We also discuss potential factors that may have contributed to this unusual bacterial infection and propose that DNA sequencing can be a useful tool in cases where standard culture techniques fail to identify the causative agent. Additionally, we highlight the importance of further research on the cryopreservation of fat. In summary, this case highlights the possibility of rare bacterial infections occurring after fat grafting procedures and emphasizes the importance of identifying the causative agent through advanced techniques such as DNA sequencing. Further research is needed to improve our understanding of the risks associated with cryopreservation of fat and to identify ways to prevent these types of infections in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 You, Yao, Gao and Liao.)

Background: Low early macrophage fat graft infiltration (within a week of surgery) hinders tissue regeneration, suggesting that macrophages play a vital role in early angiogenesis and adipogenesis. However, the source of macrophages during this period is unclear., Method: C57BL/6 mice were split into fascial removal (FR) group and control groups (CG). Mice had a piece of back fascia removed in the FR group, which was immediately replaced in the CG, and inguinal fat injected into the transplantation site of both groups. Separately, fascia was harvested from green fluorescent protein-expressing mice and transplanted into C57BL/6 mice for tracing macrophage infiltration after fat grafting., Results: The number of capillaries in the FR group was lower than that in the CG at days 3 ( P < 0.01) and 7 ( P < 0.05). Moreover, the number of small adipocytes in the FR group was lower than in the CG on days 3, 7, and 14 (all P < 0.05), and the relative expression of several adipogenic proteins was significantly lower in the FR group than in the CG on days 14 and 30. The timeline of macrophage infiltration was consistent with angiogenesis and adipogenesis. The number of macrophages in the FR group was significantly lower than in the CG at days 3 and 7 ( P < 0.05), and there were more fascia-derived macrophages than circulation-derived macrophages infiltrated into fat grafts within 7 days. Finally, the graft retention was lower in the FR group than the CG at day 90 ( P < 0.05)., Conclusion: In the early stage after fat grafting, fascial macrophage infiltration initiates tissue regeneration, thereby improving graft retention by promoting angiogenesis and adipogenesis., Clinical Relevance Statement: In the clinic, injecting fat close to the fascia may increase fat retention. Fascia is widespread and self-regenerating, which may be a promising alternative source of local macrophages, with implications for tissue-engineering therapies such as correction of soft-tissue defects and breast reconstruction., (Copyright © 2023 by the American Society of Plastic Surgeons.)

Clinical unpredictability and variability following fat grafting remain non-negligible problems due to the unknown mechanism of grafted fat retention. The role of the extracellular matrix (ECM), which renders cells with structural and biochemical support, has been ignored. This study aimed to clarify the ECM remodeling process, related cellular events, and the spatiotemporal relationship between ECM remodeling and adipocyte survival and adipogenesis after fat grafting. Labeled Coleman fat by the matrix-tracing technique was grafted in nude mice. The ECM remodeling process and cellular events were assessed in vivo. The related cytokines were evaluated by qRT-PCR. An in vitro cell migration assay was performed to verify the chemotactic effect of M2-like macrophages on fibroblasts. The results demonstrated that in the periphery, most of the adipocytes of the graft survived or regenerated, and the graft-derived ECM was gradually replaced by the newly-formed ECM. In the central parts, most adipocytes in the grafts died shortly after, and a small part of the graft-derived and newly-formed ECM was expressed with irregular morphology. Adipose ECM remodeling is associated with increased infiltration of macrophages and fibroblasts, as well as up-regulated expression of cytokines in the adipose tissue. To sum up, our results describe the various preservation mode of fat grafts after transplantation and underscore the importance of macrophage-mediated ECM remodeling in graft preservation after fat grafting. The appreciation and manipulation of underlying mechanisms that are operant in this setting stand to explore new therapeutic approaches and improve clinical outcomes of fat grafting., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: Patients with a low BMI may have inadequate high-quality adipose tissue for transplantation. The influence of high-energy diets on adipose tissue and graft retention remains unknown., Objectives: The authors explored inguinal fat pad alternation in mice fed on a short-time high-fat diet (HFD) or a high-carbohydrate diet (HCD) preoperatively and the morphological and histological differences after transplantation., Methods: Mice were fed HFD (60% kcal from fat, 20% from carbohydrate), HCD (9.3% kcal from fat, 80.1% from carbohydrate), or normal (12% kcal from fat, 67% kcal from carbohydrate) diets for 2 or 4 weeks. Histological analyses were carried out following hematoxylin and eosin staining as well as CD34 and proliferating cell nuclear antigen immunostaining. The uncoupling protein-1 expression was determined by western blotting. Fat pads from each group were grafted into the dorsal region of the recipient mice, and morphological and histological changes were determined 4, 8, and 12 weeks posttransplantation. Vascular endothelial growth factor-α and platelet-derived growth factor-α expression were determined using quantitative polymerase chain reaction., Results: The inguinal fat pad volume increased in the HFD and HCD groups. The presence of multilocular adipocytes in inguinal fat of HCD-fed mice, combined with the increased uncoupling protein-1 content, suggested adipocyte browning. HCD grafts showed higher volume retention and reduced oil cyst formation, possibly attributed to better angiogenesis and adipogenesis., Conclusions: HCD enlarged adipose tissue and improved graft survival rates, which may be due to the browning of fat before grafting and enhanced angiogenesis after grafting., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Aesthetic Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Due to the low percentage of collagen, the rigid support capacity of fat grafts remains unsatisfactory for some clinical applications. In this study, we evaluated a strategy in which adipose matrix complex (AMC) was collected via a mechanical process and transplanted for supportive filling of the face. Our AMC samples were collected from adipose tissue by a filter device consisting of a sleeve, three internal sieves, and a filter bag (100 mesh). AMC derived from adipose tissue had fewer cells than Coleman fat, but much higher levels of collagen and stiffness. Retention rates 90 days after transplantation in nude mice were higher for AMC than for Coleman fat (75±7.5% vs. 42±13.5%; P < 0.05). In addition, AMC maintained a higher stiffness (~6 kPa vs. ~2 kPa; P < 0.01) and stably retained a higher level of collagen. Our findings demonstrate that mechanical collection of AMC from adipose tissue is a practical method for improving fat graft retention and rigid support. This strategy has the potential to improve the quality of lipoaspirates for patients requiring rigid supportive filling.

Inflammatory responses mediated by macrophages play a role in tissue repair. However, it is unclear whether the repair in the donor site after liposuction would have any effects on fat graft retention in the recipient site. This study is designed to evaluate the effects of a macrophage-mediated inflammatory response in donor sites on long-term retention of fat grafting. In this study, mice were randomly divided into two groups. One underwent simulated liposuction, called the fat procurement plus grafting (Pro-Grafting) group, and the other underwent sham surgery, called the fat grafting only (Grafting Only) group. The prepared fat (0.3 ml each) was engrafted and cellular events over a 90-day period were assessed. We found macrophages were infiltrated into adipose tissue at the recipient site in the Grafting Only group within 7 days and the repair essentially completed within 30 days. By contrast, few macrophages infiltrated the recipient site in the Pro-Grafting group within 7 days and the entire remodeling process took 30 days longer in the Pro-Grafting than the Grafting Only group. Moreover, C-reactive protein levels were immediately upregulated after surgery, and the inflammatory factors' expression was higher at the donor rather than the recipient site. However, the repair processes and the long-term retention rate became normal when the adipose tissue was grafted after the donor site did not require macrophages for repair. Therefore, we suggest higher inflammatory factors promote macrophage infiltration and the adipose tissue regeneration process at the donor site. This process is delayed at the recipient site, which may affect long-term retention of fat grafts., (© 2020 Wiley Periodicals LLC.)

Background: Mature adipocytes dedifferentiate in vivo on application of a soft-tissue expander. Dedifferentiated adipocytes can proliferate and redifferentiate. This study used tissue expanders to pretreat adipose flaps, to increase the retention rate after fat graft., Methods: A soft-tissue expander and silicone sheet were implanted beneath the left and right inguinal fat pads of rats, respectively. After 7 days of expansion, the adipose tissue derived from the pads was transplanted beneath dorsal skin. Samples were harvested at various time points, and histologic, immunohistochemical, and gene expression analyses were conducted. Mature adipocytes were cultured in vitro under a pressure of 520 Pa. Changes in cell morphology, the cytoskeleton, and expression of mechanical signal-related proteins were investigated., Results: Pressure in adipose flaps increased to 25 kPa on expansion. Mature adipocytes dedifferentiated following expansion. At 1 week after transplantation, the expression of vascular endothelial growth factor (p < 0.05) was higher in the expanded group. The retention rate at 12 weeks after transplantation was higher in the expanded group (56 ± 3 percent) than in the control group (32 ± 3 percent) (p < 0.05), and the surviving/regenerating zones (p < 0.01) were wider. The lipid content of mature adipocytes gradually decreased on culture under increased pressure, and these cells regained a proliferative capacity. This was accompanied by increased expression of mechanical signal--related proteins (p < 0.05)., Conclusions: Mechanical signals may induce dedifferentiation of mature adipocytes. Dedifferentiated adipocytes increase the retention rate of fat grafts by acting as seed cells.

Mesenchymal stem cells reportedly have a marked effect on tumor growth or suppression. However, it remains uncertain whether adipose‑derived mesenchymal stem cells (ADSCs) from grafted fat can contribute to breast cancer growth and recurrence. In the present study, interactions between ADSCs and MCF‑7 breast cancer cells were evaluated in a Matrigel co‑culture system and in an in vivo nude mouse model. Results suggested that MCF‑7 cells exerted tumor tropism effects on ADSCs and this may be regulated by chemokines, such as the macrophage inflammatory protein (MIP)‑1δ and MIP‑3α. Additionally, ADSCs significantly induced tumorsphere formation in vitro and promoted tumorigenicity in vivo. RT‑qPCR analysis indicated that tumorsphere formation by MCF‑7 cells was associated with the induction of stem‑like properties, which was mediated by epithelial‑-mesenchymal transition. Together, the present findings indicated that ADSCs exhibit tropism and induce tumorsphere formation of MCF‑7 cells.

Background: External volume expansion devices are effective for adipose tissue regeneration. However, the detailed mechanisms by which external volume expansion devices induce adipose tissue regeneration remain unclear., Methods: An external volume expansion device was used to construct expanded prefabricated adipose tissue in a rat model. CXCL12 levels in local exudate and serum were measured by enzyme-linked immunosorbent assay, and CXCL12 expression in adipose tissue was assessed immunohistochemically. Fluorescent dye (CM-DiI)-labeled bone marrow-derived mesenchymal stromal cells and labeled mesenchymal stromal cells pretreated with the CXCR4 antagonist AMD3100 were transplanted into rats and tracked in vivo by fluorescence imaging., Results: CXCL12 levels in local exudate and serum peaked 2 and 7 days, respectively, after external volume expansion device application. CXCL12 cell counts were significantly higher in the external volume expansion than in the control group. These CXCL12 cells were mainly columnar or cuboidal and began to express peroxisome proliferator-activated receptor γ on day 9. CM-DiI-labeled mesenchymal stromal cells were successfully recruited to the expanded prefabricated adipose tissue, a process partly inhibited by the CXCR4 antagonist AMD3100. These recruited CM-DiI-labeled mesenchymal stromal cells were found among the CXCL12 columnar cells., Conclusions: External volume expansion devices enhance CXCL12 expression levels, especially in columnar and cuboidal cells. The CXCL12/CXCR4 pathway is involved in recruiting circulating mesenchymal stromal cells to participate in adipose regeneration. These findings may reveal the mechanism underlying external volume expansion and provide insights into the refinement of these devices.

Background: Flap necrosis due to insufficient blood supply is a common postoperative complication in random pattern flaps. Stem cell therapies have emerged as promising biologics for tissue ischemia. A novel fat derived product, stromal vascular fraction gel (SVF-gel), can be prepared with lipoaspirate through simple mechanical processing, removing only the lipid content. SVF-gel enriches adipose-derived stem cells and potentially beneficial for flap necrosis., Methods: Nude mice ischemic flaps were treated with human SVF-gel, stromal vascular fraction (SVF) cell suspension or saline (n = 10). They were injected to the flap recipient beds, and necrosis and vascularization was assessed on postoperative day 14. We harvested the necrosis-free distal to evaluated skin healthiness and neovasculogenesis by Masson's trichrome stain and immunofluorescence, etc. Pro-angiogenic factors were assessed with tissue qRT-PCR. Finally, we traced the grafted human tissue with immunofluorescence., Results: SVF-gel-treated flaps have the smallest necrotic zones (22.05% ± 0.0438) compared with the saline controls (53.78% ± 0.1412) or SVF-treated ones (35.54% ± 0.0850, p = 0.039). Numerous functional musculocutaneous perforators were developed around SVF-gel grafts. The SVF-gel-treated skin had the best fat restoration (231.3 ± 48.1 μm) among three groups (F = 10.83, p = 0.0102) while saline-treated flap distal appeared fibrotic. SVF-gel-treated flaps also had ∼43% more CD31  +  capillaries (p = 0.0152) with ∼3 folds more gene expression of angiogenic cytokines of VEGF and bFGF (p = 0.0310 and 0.0303, respectively) than saline-treated controls. Furthermore, we found hSVF-gel cells (hGolgi+) had directly engrafted as vessel component (α-smooth muscle actin, α-SMA+) to the flap., Conclusion: Adipose cellular matrix enhanced flap neovascularization partly by direct incorporation, improved flap survival and fat restoration. The composition-selective fat grafting with SVF-gel demonstrated efficacy comparable with stem cell therapy and is especially valuable for clinical translation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Mature adipocytes can dedifferentiate into fibroblast-like cells in vitro and acquire proliferation and redifferentiation/transdifferentiation abilities. A soft-tissue expander can induce adipocyte dedifferentiation in vivo. This study combined a tissue expander and an external suspension device to generate a large volume of adipose tissue., Methods: A soft-tissue expander was implanted beneath the dorsal adipose flaps of rabbits. After 7 days of expansion, the expander was removed and an external suspension device was applied. Samples were collected at various time points, and morphologic, histologic, immunohistochemical, and gene expression analyses were conducted. A silicone sheet was implanted as a control., Results: After 7 days of expansion, the adipose flap was much thinner. Hematoxylin and eosin and whole-mount staining revealed that adipocytes became smaller (p < 0.05) and some contained multilocular lipid droplets. The number of Ki67 cells increased (p < 0.01), adipokine expression decreased (p < 0.01), and octamer-binding transcription factor 4 expression increased (p < 0.01). After the external suspension device was applied, the normalized volume of adipose flaps was much larger in the expanded group than in the control group (p < 0.05). The expanded group also exhibited more proliferating cells, a larger vascularized area, and higher adipokine expression (p < 0.05)., Conclusion: Dedifferentiated adipocytes in adipose flaps can participate in adipose tissue generation as seed cells and increase the volume of adipose tissue.

Background: Multiple studies have demonstrated that mechanical forces promote the growth of adipose tissue. However, the mechanism of adipose tissue regeneration induced by mechanical forces remains unclear., Methods: In an experiment using rats, prefabricated adipose tissue with a vessel pedicle was expanded using an external volume expansion device. The volume of fat flaps was tested at different time points. Cell proliferation and angiogenesis were analyzed using immunofluorescence. The expression of adipogenic genes and inflammatory cytokines was evaluated using real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay, respectively., Results: There were more CD31 cells and Ki67/CD34 cells in the experimental group than in the control group. The number of Ki67/CD34 cells peaked at 1 to 4 weeks. However, the expression levels of peroxisome proliferator-activated receptor-γ and CCAAT/enhancer-binding protein-β were highest from 4 to 12 weeks in the experimental group. Compared with the control group, the experimental group showed more proinflammatory cytokines: interleukin-1β, interleukin-6, tumor necrosis factor-α, and macrophage migration inhibitory factor., Conclusions: The construction of expanded prefabricated adipose tissue by mechanical forces is a dynamic and complex process. Mechanical forces promoted cell proliferation and angiogenesis in the early stage of adipose tissue regeneration (before 4 weeks) and induced adipogenic differentiation at a later stage (after 4 weeks) through up-regulation of macrophage migration inhibitory factor, which provided an adipogenic inductive microenvironment.

Background: Adipose-derived stem cells and other stromal vascular fraction cells were used more often for stem cell therapy, even though limitations such as poor cell retention rate, complicated and expensive isolation processes, and the use of specific laboratory equipment need to be overcome., Methods: Here, the authors developed a novel but simple method for generating an injectable mixture of stromal vascular fraction cells and native adipose extracellular matrix. It is a purely mechanical process in which lipoaspirate is processed into an extracellular matrix/stromal vascular fraction gel. The standard processing procedure was established using quantized tests. The therapeutic potential of the product for wound healing was then tested., Results: Extracellular matrix/stromal vascular fraction gel derived from lipoaspirate and processed using a standard Coleman technique, followed by 1 minute of mechanical processing by passage back and forth between two 10-ml syringes at a flow rate of 10 ml/second, showed the highest adipose-derived stem cell and endothelial cell density. The stromal vascular fraction cells within the product also showed potential for multipotent differentiation similar to that of normal fat samples. In addition, the product showed better therapeutic results than stromal vascular fraction cell suspension when used to treat a nude mouse model of wound healing., Conclusions: Extracellular matrix/stromal vascular fraction gel is an autologous injectable derived from native extracellular matrix and is a functional cellular component generated using a simple mechanical process. As such, it may offer a novel mode of tissue repair suitable for clinical application in stem cell therapies.

Background: Fat grafting has variable and sometimes poor outcomes, and therefore new methods are needed. Multiple studies have demonstrated the excellent performance of external volume expansion and focused only on preexpansion with emphasis on the recipient., Methods: Two mouse models (a suction model and a fat-exchange transplantation model) were established to investigate changes in the origins and biological behaviors of regeneration-related cells in grafted fat under daily suction provided by external volume expansion., Results: Blood supply increased from new host-derived capillaries or macrophage infiltration under suction. CD34-positive cells showed increased migration from the host into the grafts under suction. At week 12, nearly half of the mature adipocytes regenerated in the grafts in the suction group were derived from the host. Peroxisome proliferator-activated receptor γ expression of the suction group was significantly higher than that of controls at weeks 2 and 4 during adipogenesis. The normalized sample weight of the grafted fat was significantly greater than that of controls at 1 (0.081 ± 0.001 versus 0.072 ± 0.005; p < 0.001), 4 (0.060 ± 0.002 versus 0.048 ± 0.001; p = 0.002), 8 (0.060 ± 0.001 versus 0.046 ± 0.001; p < 0.001), and 12 weeks (0.060 ± 0.001 versus 0.046 ± 0.001; p = 0.002)., Conclusions: The mechanical effect of daily suction provided by external volume expansion favors the regeneration of grafted fat and improves retention by promoting the migration of regeneration-related cells and the differentiation of adipocytes. Thus, more mature fat tissue with a well-organized structure was formed under suction.

Mesenchymal stem cells are an attractive cell type for cytotherapy in wound healing. The authors recently developed a novel, adipose-tissue-derived, injectable extracellular matrix/stromal vascular fraction gel (ECM/SVF-gel) for stem cell therapy. This study was designed to assess the therapeutic effects of ECM/SVF-gel on wound healing and potential mechanisms. ECM/SVF-gel was prepared for use in nude mouse excisional wound healing model. An SVF cell suspension and phosphate-buffered saline injection served as the control. The expression levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and monocyte chemotactic protein-1 (MCP-1) in ECM/SVF-gel were analyzed at different time points. Angiogenesis (tube formation) assays of ECM/SVF-gel extracts were evaluated, and vessels density in skin was determined. The ECM/SVF-gel extract promoted tube formation in vitro and increased the expression of the angiogenic factors VEGF and bFGF compared with those in the control. The expression of the inflammatory chemoattractant MCP-1 was high in ECM/SVF-gel at the early stage and decreased sharply during the late stage of wound healing. The potent angiogenic effects exerted by ECM/SVF-gel may contribute to the improvement of wound healing, and these effects could be related to the enhanced inflammatory response in ECM/SVF-gel during the early stage of wound healing.

An adipose tissue engineering chamber model of spontaneous adipose tissue generation from an existing fat flap has been described. However, the chamber does not completely fill with adipose tissue in this model. Here, the effect of adipose tissue extract (ATE) on adipose tissue regeneration was investigated. In vitro, the adipogenic and angiogenic capacities of ATE were evaluated using Oil Red O and tube formation assays on adipose-derived stem cells (ASCs) and rat aortic endothelial cells (RAECs), respectively. In vivo, saline or ATE was injected into the adipose tissue engineering chamber 1 week after its implantation. At different time points post-injection, the contents were morphometrically, histologically, and immunohistochemically evaluated, and the expression of growth factors and adipogenic genes was analyzed by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR. With the exception of the baseline control group, in which fat flaps were not inserted into a chamber, the total volume of fat flap tissue increased significantly in all groups, especially in the ATE group. Better morphology and structure, a thinner capsule, and more vessels were observed in the ATE group than in the control group. Expression of angiogenic growth factors and adipogenic markers were significantly higher in the ATE group. ATE therefore significantly promoted adipose tissue regeneration and reduced capsule formation in an adipose tissue engineering chamber model. These data suggest that ATE provides a more angiogenic and adipogenic microenvironment for adipose tissue formation by releasing various cytokines and growth factors that also inhibit capsule formation.

Background: Although fat is transplanted into several layers, including subcutaneous, fat, and muscle layers, there is little (clinical) scientific basis for these procedures., Objective: To determine the optimal recipient layer for fat transplantation., Materials and Methods: Fat harvested from inguinal fat pads of green fluorescent protein (GFP) mice was grafted into the subcutaneous and intramuscular planes and the fat pads of C57 mice. Specimens collected after 1, 4, 8, 12, and 16 weeks were stained with hematoxylin and eosin to determine angiogenesis and fibrosis in the grafts. The survival rate of donor adipose tissue was determined by measuring GFP expression., Results: Fat was retained longer in fat pads than in subcutaneous layers of recipient mice and longer in subcutaneous than in intramuscular layers. Angiogenesis and vascularized connective tissue were greater in intramuscular than in subcutaneous or fat grafts. Neovascularization, however, was similar in fat pads and subcutaneous grafts. Survival rate was higher for intramuscularly injected fat than subcutaneously and fat pad injected fat., Conclusion: Fat pad injection showed the highest graft retention rate, indicating that fat pads maybe the optimal area for fat transplantation. The increased blood supply to muscle suggests that intramuscular injection maybe optimal when there is little movement.

In plastic and reconstructive surgery, adipose tissue is widely used as effective filler for tissue defects. Strategies for treating soft tissue deficiency, which include free adipose tissue grafts, use of hyaluronic acid, collagen injections, and implantation of synthetic materials, have several clinical limitations. With the aim of overcoming these limitations, researchers have recently utilized tissue engineering chambers to produce large volumes of engineered vascularized fat tissue. However, the process of growing fat tissue in a chamber is still relatively limited, and can result in unpredictable or dissatisfactory final tissue volumes. Therefore, detailed understanding of the process is both necessary and urgent. Many studies have shown that mechanical force can change the function of cells via mechanotransduction. Here, we hypothesized that, besides the inflammatory response, one of the key factors to control the regeneration of vascularized fat flap inside a tissue engineering chamber might be the balance of mechanical forces. To test our hypothesis, we intend to change the balance of forces by means of measures in order to make the equilibrium point in favor of the direction of regeneration. If those measures proved to be feasible, they could be applied in clinical practice to engineer vascularized adipose tissue of predictable size and shape, which would in turn help in the advancement of tissue engineering., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Advances in structural fat transplantation technology have significantly improved the survival rate and stability of grafts. This study investigated the importance of the spatial structural integrity of adipose tissue for adipose regeneration after fat transplantation. We sought to enhance understanding of structural fat transplantation and optimize procedures used for the clinical acquisition, purification, and transplantation of adipose tissue. In an inactivated structuration adipose tissue model established by freezing at -20 °C for 3 days, nearly all cells were dead but the structure was intact. We transplanted this adipose tissue model (group A) or non-treated adipose tissue (group B) into GFP-expressing mice. Group B showed a higher graft survival percentage and less fibrosis than group A. The macrophage infiltration (F4/80) peak period was longer in group A than in group B. The change in vessel density (CD31) was similar in the two groups: it peaked at 4 weeks after transplantation and decreased thereafter. In both groups, the number of Ki67+ cells showed a similar trend. In comparison to group B, group A had more Ki67+ cells at 4-8 weeks after transplantation, but fewer of these cells at 12 weeks after transplantation. The intact spatial structure of adipose tissue, which is supported by adipocytes and extracellular matrix, provides a niche for adipogenesis and angiogenesis after fat transplantation.

Objective: To evaluate the influence of adipose tissue extract on inducing angiogenesis and adipogenesis in adipose tissue engineering chamber in vivo., Methods: 6 months' healthy New Zealand rabbits (n = 64) were picked. The inguinal fat pads were cultured, centrifuged, filtered, and the liquid was called adipose tissue extract (ATE). Two adipose tissue engineering chamber were built in the rabbit's back. A week later, 0.2 ml normal saline (control group, left) and 0. 2 ml ATE (experimental group, right) was respectively injected into the chamber. The contents were evaluated morphometrically, histologically and immunohistochemically 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks and 7 weeks after injection. 8 rabbits were observed each time. The data regarding the number of the volume of fat flap and blood capillary at each time point were analyzed by paired t test., Results: After injection, new tissue volume was significantly increased in the experimental group [(5.12 ± 0.22) ml], compared with that in control group [(4.90 ± 0.15) ml]. Early angiogenesis was also increased after ATE injection and the total number of capillaries reached peak 1 week after injection, which was (72.80 ± 9.67) in experimental group and (51.40 ± 6.09) in control group. In the mid-term of experimental period, earlier adipogenesis appeared in experimental group. In the later period, the outer capsule of the new construction was thinner in experimental group which reduced the suppression of the adipogenesis., Conclusions: ATE can promote the angiogenesis and adipogenesis in the chamber, and reduce the capsule contracturing, so as to induce the large volume of adipose tissue regeneration

The development of an engineered adipose tissue substitute capable of supporting reliable, predictable, and complete fat tissue regeneration would be of value in plastic and reconstructive surgery. For adipogenesis, a tissue engineering chamber provides an optimized microenvironment that is both efficacious and reproducible; however, for reasons that remain unclear, tissues regenerated in a tissue engineering chamber consist mostly of connective rather than adipose tissue. Here, we describe a chamber-based system for improving the yield of mature adipose tissue and discuss the potential mechanism of adipogenesis in tissue-chamber models. Adipose tissue flaps with independent vascular pedicles placed in chambers were implanted into rabbits. Adipose volume increased significantly during the observation period (week 1, 2, 3, 4, 16). Histomorphometry revealed mature adipose tissue with signs of adipose tissue remolding. The induced engineered constructs showed high-level expression of adipogenic (peroxisome proliferator-activated receptor γ), chemotactic (stromal cell-derived factor 1a), and inflammatory (interleukin 1 and 6) genes. In our system, the extracellular matrix may have served as a scaffold for cell migration and proliferation, allowing mature adipose tissue to be obtained in a chamber microenvironment without the need for an exogenous scaffold. Our results provide new insights into key elements involved in the early development of adipose tissue regeneration., (© 2015 by the Wound Healing Society.)

Introduction: Adipocytes can dedifferentiate into fibroblast-like cells in vitro and thereby acquire proliferation and multipotent capacities to participate in the repair of various organs and tissues. Whether dedifferentiation occurs under physiological or pathological conditions in vivo is unknown., Methods: A tissue expander was placed under the inguinal fat pads of rats and gradually expanded by injection of water. Samples were collected at various time points, and morphological, histological, cytological, ultrastructural, and gene expression analyses were conducted. In a separate experiment, purified green fluorescent protein+ adipocytes were transplanted into C57 mice and collected at various time points. The transplanted adipocytes were assessed by bioluminescence imaging and whole-mount staining., Results: The expanded fat pad was obviously thinner than the untreated fat pad on the opposite side. It was also tougher in texture and with more blood vessels attached. Hematoxylin and eosin staining and transmission electron microscopy indicated there were fewer monolocular adipocytes in the expanded fat pad and the morphology of these cells was altered, most notably their lipid content was discarded. Immunohistochemistry showed that the expanded fat pad contained an increased number of proliferative cells, which may have been derived from adipocytes. Following removal of the tissue expander, many small adipocytes were observed. Bioluminescence imaging suggested that some adipocytes survived when transplanted into an ischemic-hypoxic environment. Whole-mount staining revealed that surviving adipocytes underwent a process similar to adipocyte dedifferentiation in vitro. Monolocular adipocytes became multilocular adipocytes and then fibroblast-like cells., Conclusions: Mature adipocytes may be able to dedifferentiate in vivo, and this may be an adipose tissue self-repair mechanism. The capacity of adipocytes to dedifferentiate into stem cell-like cells may also have a more general role in the regeneration of many tissues, notably in fat grafting.

Our objective was to explore the mechanism of cell-assisted adipose transplantation by using freshly isolated human stromal vascular fraction (SVF) cells and to observe the dynamic changes of the graft after transplantation. The SVF was isolated from human liposuction aspirates, and 0.5 ml adipose tissue was mixed with 1 × 10(6) SVF cells or culture medium then injected to nude mice subcutaneously. At 1, 4, 7, 14, 30, 60, and 90 days after transplantation, samples were harvested for 1) general observation and retention rate; 2) whole-mount stain; 3) H&E stain; 4) immunohistochemical staining for S100, CD68, and CD34; 5) ELISA for VEGF and bFGF; 6) peroxisome proliferator-activated receptor-γ (PPARγ) fluorescence in situ hybridization. The retention rate in the experiment group was markedly higher than that in the control group. Whole-mount stain shows most of the cells in the center of the graft could not survive the ischemia until day 14. Histology showed new vessels on the surface of the graft at 3 days. However, in the control group, fewer newly formed vessels were detected until day 7. In the late stage of transplantation, gradual fibrosis was found in the graft, and the tissue was divided into a grid-like structure. A large number of round neonatal adipocytes with big nuclei in the center were found surrounding the new vessels, which were S100 and CD34 positive and CD68 negative. In the late stage of transplantation, most of the neonatal adipocytes were human PPARγ positive. Moreover, the SVF group showed a higher level of VEGF and bFGF. SVF assisting adipose transplantation could increase the retention rate of the graft through promoting adipose tissue regeneration via secretion of growth factors, promotion of angiogenesis, and increasing the density of mesenchymal stem cells.

Liposuction aspirates separate into fatty and fluid portions. Cells isolated from the fatty portion are termed processed lipoaspirate (PLA) cells and isolated from the fluid portion termed liposuction aspirate fluid (LAF) cells, both of which contain adipose-derived stromal cells (ASCs). Here, we examined the biological differences between PLA and LAF cells and then tested the differentiation capacity of LAF cells in vivo. The cell surface marker and the multiple differentiation ability of fresh isolated PLA and LAF cells and which from passaged 3-5 were examined in vitro. LAF cells were then incubated in adipogenic medium, stained with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI), mixed with fibrin glue then injected to nude mice; fibrin glue without cells was as a control. Three months later, the transplants were subjected to macroscopic observation and histological analysis. PLA and LAF cells were similar in growth kinetics, morphology, capacity for differentiation, and surface marker profiles. After plating, both PLA and LAF cells showed increased expression of CD29, CD44, CD133 and HLA DR and decreased expression of CD34. In vivo differentiation assay showed the mixture of LAF cells and fibrin glue formed adipose tissue which contained red fluorescent DiI-positive adipocytes. LAF cells can be harvested more easily than PLA cells. The in vivo adipogenic capacity suggested LAF cells would be useful and valuable for cell-based therapies and soft tissue reconstruction., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Mesenchymal stem cells (MSCs) are adult multipotent cells found in bone marrow, adipose tissue, and other adult tissues. MSCs improve regeneration of injured tissues in vivo, but the mechanisms remain unclear. Typically, MSCs are cultured under ambient or normoxic conditions (21% O2 ). However, the physiological niches of MSCs have much lower oxygen tension. When used as a therapeutic tool to repair tissue injuries, MSCs cultured in standard conditions must adapt from 21% O2 in culture to <1% O2 in ischemic tissue. We have examined the effects of hypoxia preconditioning (1% O2 ) in human adipose derived mesenchymal stem cells (AD-MSCs) to discover the conditions that best enhance their tissue regenerative potential. We demonstrate that AD-MSCs respond positively to hypoxia compared with normoxia preconditioning, show decreased apoptosis even in severe microenvironmental conditions (such as a low-serum medium), and an increased expression of the angiogenic factors, vascular endothelial growth factor and basic fibroblast growth factor. Human umbilical vein endothelial cells have higher vitality and lower apoptosis when cultured in medium taken from hypoxia-preconditioned AD-MSCs, as well as significantly increased capillary-like structures with this medium on Matrigel. The data suggest that hypoxia preconditioned AD-MSCs can improve tissue regeneration., (© 2013 International Federation for Cell Biology.)

Objective: To investigate the mechanism of vascular stromal fraction (SVF) at the early stage after aspirated fat transplantation., Methods: Fat was harvested from 5 cases of women undergoing abdominal liposuction operation, and SVF was isolated. Aspirated fat with (group B) or without (group A) SVF was injected subcutaneously into the back of nude mice, and the grafts were harvested at 1, 3, 5, and 7 days. Graft wet weight was measured; and immunohistochemical method (CD31) was performed and the secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were qnantified by Western blot assay., Results: The wet weight of transplanted adipose tissue showed an increasing tendency in groups A and B with time, and no significant difference was found between groups A and B (P > 0.05). At 1 and 3 days after transplantation, no CD31 positive cells was seen in 2 groups; the CD31 positive cells of group B were significantly more than those of group A at 5 and 7 days (P < 0.05), and the CD31 positive cells at 7 days were significantly more than those at 5 days in 2 groups (P < 0.05). Western blot test showed that VEGF expression reached peak at 3 days , then decreased gradually; the expression of VEGF protein in group B was significantly higher than that in group A at 1, 3, and 5 days (P < 0.05). The expression of HGF protein in groups A and B remained at a high level within 5 days, but it tended to decrease at 7 days, which was significantly higher in group B than that in group A (P < 0.05)., Conclusion: SVF can enhance angiogenesis by secretion of growth factors at the early stage after aspirated fat transplantation.

Background: Numerous methods have been proposed to enhance the survival of fat grafts, but no definitive treatment is available. Stromal vascular fraction (SVF)-assisted cell therapy offers new perspectives for improving fat graft survival., Objectives: To determine whether SVF supplementation could improve graft retention in patients undergoing autologous fat grafting for cosmetic improvement of facial contour., Methods: We retrospectively analyzed data from 38 women who underwent fat transplantation with SVF (n = 26) or fat grafting alone (n = 12) between October 2010 and January 2012. Each patient underwent computed tomography, and photographs were taken before and 6 months after surgery. The Philips Extended Brilliance Workspace was used for analysis of volume augmentation., Results: All patients showed cosmetic improvements, but the degree varied. No complications were evidenced during follow-up. Fat survival was higher with SVF (64.8 ± 10.2%) than fat grafting alone (46.4 ± 9.3%) (p < .01). SVF supplementation resulted in better clinical improvement than fat grafting alone., Conclusion: Supplementing fat grafts with SVF for cosmetic facial contouring can improve the survival of fat grafts over fat grafting alone and provides satisfactory outcomes without major complications. Autologous fat grafting has been used for various cosmetic treatments and difficult reconstructive indications such as temporal depression, wrinkles of nasolabial folds, and hemifacial atrophy, with no incisional scar or complications associated with foreign materials, although problems such as a low rate of graft survival because of early resorption remain. (Aesthet Plast Surg, 14, 1990 and 127) Despite many innovations to overcome these problems, (Dermatol Surg, 26, 2000 and 1159); (Ann Plast Surg, 60, 2008 and 594); (Dermatol Surg, 27, 2001 and 819); (Dermatol Surg, 28, 2002 and 987) we lack a definitive method of fat processing that ensures maximal graft take and viability. (Plast Reconstr Surg, 115, 2005 and 197); (Dermatol Surg, 37, 2011 and 619)., (© 2012 by the American Society for Dermatologic Surgery, Inc. Published by Wiley Periodicals, Inc.)

Background: Autologous fat grafting has rapidly become an important treatment for soft tissue defects in cosmetic and reconstructive surgery. However, consensus is lacking on the ideal donor site for harvesting and isolating stromal vascular fraction (SVF) cells to improve survival of fat grafts. We aimed to determine the best donor site for tissue harvesting and isolation of SVF cells for fat graft survival., Methods: Adipose tissue samples were harvested from six women who underwent an aesthetic procedure. The samples were harvested by needle aspiration of five commonly used donor sites: flank, upper and lower abdomen, and lateral and inner thigh. The adipose tissue was injected subcutaneously into nude mice and grafts were harvested at 12 weeks. We evaluated graft volume, weight, and histologic parameters of the grafts: integrity, cysts/vacuoles, inflammation, fibrosis, and neovascularization. SVF cells isolated from donor sites were counted and assayed by flow cytometry., Results: At 12 weeks post-transplantation, weight, volume, and histologic parameters did not differ among the grafts from the five tissue donor sites. Also, SVF and levels of cell surface markers did not differ by donor site., Conclusions: This study revealed no ideal tissue donor site for fat grafting and SVF isolation. Choosing a site should be based on ease and safety of access and the preference and request of the patient., Level of Evidence I: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Objective: To review the mechanism of improved revascularization of free fat grafting with adipose-derived stem cells (ADSCs)., Methods: The literature related to the basic researches of ADSCs in free fat grafting and angiogenesis was reviewed., Results: Angiogenesis is a sequence process in time and space which is regulated by various factors. ADSCs possess the capability of secreting many angiogenic growth factors and differentiating into various lineages., Conclusion: ADSCs affect every process of angiogenesis with clear improved angiogenic effects, however, the mechanisms of angiogenic effects need the further researches.

Background: The efficacy of autologous fat transplantation is reduced by fat absorption and fibrosis due to fat necrosis. Enhanced transplant neovascularization early after transplantation may reduce these outcomes. The authors asked whether cell and concomitant gene therapy using adipose-derived stem cells transduced with vascular endothelial growth factor (VEGF) improves fat transplant neovascularization and survival., Methods: Human adipose-derived stem cells were expanded ex vivo for three passages, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI), and transduced with VEGF or left untransduced. Human fat tissues were then mixed with the DiI-labeled VEGF-transduced adipose-derived stem cells, the DiI-labeled adipose-derived stem cells, the known vascularization-promoting agent insulin, or medium alone, and 18 nude mice were injected subcutaneously with all four preparations, with each of the four designated spots receiving one of these four mixtures in a random fashion. Six months later, transplanted tissue volume and histology were evaluated and neovascularization was quantified by counting the capillaries., Results: Control transplant survival was 27.1 +/- 8.2 percent, but mixture with the VEGF-transduced and VEGF-untransduced stem cells significantly increased transplant survival (74.1 +/- 12.6 percent and 60.1 +/- 17.6 percent, respectively). Insulin was less effective (37.7 +/- 6.9 percent). Histological analysis revealed both types of transplants consisted predominantly of adipose tissue, unlike the control transplants, and had significantly less fat necrosis and fibrosis. The VEGF-transduced, adipose-derived stem cell-treated transplants had significantly higher capillary density than the other transplants and bore DiI-double-positive and CD31-double-positive cells (i.e., adipose-derived stem cell-derived endothelial cells)., Conclusion: Adipose-derived stem cells together with VEGF transduction can enhance the survival and quality of transplanted fat tissues.

Objective: To compare two kinds of myofascial flap encapsulating adipose-derived stromal cells (ADSCs) in adipogenic efficacy in vivo, and to provide experimental basis for the efficient transplantation of free adipose tissue., Methods: ADSCs were isolated from the subcutaneous adipose tissue in the neck of 10 New Zealand rabbits (aged 3- 4-months-old, male and female, weighing 2.0-2.5 kg), and primary culture and subculture of ADSCs were conducted. When the cells at passage 3 covered 70%-80% of the bottom of the culture flask, BrdU (10 microg/mL) was applied to label the cells for 48 hours before performing immunofluorescence staining. Oil red O staining observation was conducted to those cells 2 weeks after being induced towards adipocyte, alizarin red staining observation was performed 3 weeks after being induced towards osteoblast, and alcian blue staining was conducted 2 weeks after being induced towards chondrocyte. Besides, after being induced towards adipocyte for 2 weeks, 1 x 10(7) ADSCs/piece at passage 3 labeled by BrdU was seeded into Col I (10 mm x 10 mm x 5 mm/piece) to prepare cell carrier complex. The experiment was divided into two groups: group A in which vascular pedicled dextral latissimus dorsi fascial flap was adopted to encapsulate the complex; group B in which dextral gluteus maximus fascial flap with no specific vessel pedicle was applied to encapsulate the complex. Rabbits in each group went through autogenous ADSCs transplant and self control. The implants were dislodged 8 weeks after operation, HE staining and immunohistochemistry staining were performed to testify cambium, the wet weight and micro vessel count of the cambium in each group were tested, immunofluorescence staining was performed to determine the origin of cambium and microvascular endothelium., Results: The nucleus of ADSCs positive for BrdU labeling showed green fluorescence under fluorescence microscope, with the positive labeling ratio of ADSCs above 90%. For ADSCs at passage 3, the formation of red lipid droplets within cells was observed 2 weeks after being induced towards adipocyte, red calcium nodules were evident 3 weeks after being induced towards osteoblast, and highly congregated cell mass positive for alcian blue staining appeared 2 weeks after being induced towards chondrocyte. Eight weeks after operation, neogenetic blood vessel grew into scaffolds and no obvious fibre encapsulation was observed in group A, while few blood vessel grew into scaffolds in group B. The wet weight of cambium in group A and B was (0.149 5 +/- 0.017 3) g and (0.095 3 +/- 0.012 7) g, respectively, indicating there was a significant difference between two groups (P < 0.01). HE staining showed the formation of neogenetic adipose tissue and the growth of micrangium in the implant, and the degradation and absorption of scaffold. The micro vessel count of group A and B was 31.2 +/- 4.5 and 19.3 +/- 2.6, respectively, indicating there was a significant difference between two groups (P < 0.01). Eight weeks after operation, the immunofluorescence staining of cambium showed that the cell nucleus of regenerated adipocytes and partial capillary endothelium in groups A and B presented green fluorescence., Conclusion: ADSCs encapsulated by vascular pedicled latissimus dorsi fascial flap and collagen protein scaffold complex has a higher adipogenic efficacy in vivo than the gluteus maximus fascial flap with no specific vessel pedicle.

Objective: To investigate the clinical effect of transplanting by auto-fat granule injection for mastatrophy post suckling., Methods: From March 2000 to June 2006, 73 patients (146 breasts) with mastatrophy post suckling were treated by transplanting auto-fat granule. The mastatrophy occurred between ages 28 and 52 years with a median of 37 years post suckling. The breasts shrank and their elasticity decreased gradually within 2-10 years post suckling. The auto-fat granule was obtained by liposuction with syringe from patient's abdomen, waist, buttocks and thighs, etc. After repeated wash and purification, the auto-fat granule was transplanted into the interspace behind the breast by injection. The quantity of auto-fat granule was 50-100 mL in each side of breast per transplantation at 3-6 months intervals, and the whole course of treatment needed 2-6 transplantations., Results: The incisions in all cases healed primarily postoperatively. In 73 cases, 65 were followed up from 6 months to 3 years post operation. All patients had a significant improvement in their breast size and shape postoperatively and their breasts were soft and natural in appearance and feel. All of them had more perfect arcuation of physique and body with strengthened self-confidence, relieved mood and improved quality of life. However, small indurations were found sporadically in 7 cases (10 breasts) within 2-7 months, and calcifications in 5 cases (8 breasts) within 9-14 months post the first operation., Conclusion: The transplantation by auto-fat granule injection for mastatrophy post suckling is an effective and practical method. The surgical technique is well worth performing in clinical practice.

Objective: To explore the optimal dosage, timing and cytotoxicity of bromodeoxyuridine (BrdU) labelling for rabbit adipose-derived stromal stem cells (ADSCs) in vitro so as to confirm its feasibility for stem cells labelling and tracer means., Methods: Six rabbits were used in this experiment, aged 8-12 weeks, weighing 1.5-2.0 kg and neglecting their gender. 1-2 mL fat was removed, the ADSCs were isolated and cultured using the adherence method in vitro. The 3rd passage of ADSCs was incubated withBrdU at 5, 10, 15 and 20 microg/mL (groups A, B, C and D) for 12, 24, 48 and 72 hours to identify the optimal BrdU concentration and incubating time for cell labelling. Immunohistochemistry and trypanblau strain were performed respectively to calculate the labelling index (positive rate) and the cells' activity for different time after BrdU labelling. The ADSCs without BrdU labelling were used as control (Group E)., Results: The main appearance of primary ADSCs was short fusiform shape, and of the 3rd passage ADSCs long fusiform shape. The 3rd passage of ADSCs could differentiate into osteoblasts and adipocytes under corresponding inductive medium. The ADSCs' nucleus show green fluor under fluorescence microscope after labeled by the BrdU. The labelling ratio increased in groups A, B, C and D after incubating 12 hours, the mean labelling ratio were 30.6% +/- 2.3%, 32.4% +/- 1.9%, 45.8% +/- 1.8%, 50.8% +/- 3.1%, respectively, and the labelling ratio of Group E was 0. There were significant differences between groups C, D and Group A (P < 0.01). The labelling ratio of groups A, B, C and D were 45.9% +/- 2.0%, 87.9% +/- 3.3%, 90.6% +/- 2.9%, 91.7%+/- 3.2%, respectively after 24 hours and the labelling ratio of Group E was 0. There were significant differences between groups B, C, D and Group A (P < 0.01). The results of all groups after incubating 48 hours and 72 hours were similar to that after incubating 24 hours. The cell counting of groups A, B, C and D were better than that of Group E, but showing no siginificant differences (P > 0.05)., Conclusion: The most appropriate time for BrdU labelling ADSCs is 48 hours, the most appropriate concentration is 10 microg/mL. The labelling rate is high and cytotoxicity is little.