Fat Regeneration Research Articles

Regenerated Fat Induced by a Decellularized Adipose Matrix Can Survive Long-term In Vivo

Decellularized adipose matrix (DAM) is considered to be the most potential biological scaffold for soft tissue repair and reconstruction, as it is able to induce the regeneration of adipose tissue in situ in adulthood. But how does this adipose tissue regeneration happen and develop in vivo? Is it the same as the original autologous one? Temporary existence or long-term survival? These are the key questions that will determine the future applications of DAM. In this study, we investigated the composition, structure and biomechanical properties of DAM before implanting it into the subcutaneous back of immunodeficient mice. The entire regeneration process in vivo was closely monitored histologically from 3 days to 1 year after implantation, including fat regeneration, vascular growth, inflammatory responses, and matrix degradation and remodeling. Transcriptome sequencing was used to analyze the difference in gene expression between regenerated fat and autologous fat at different periods. The results showed that the DAM-induced regenerated fat first appeared at 1w and remained stable over 6m, indicating remarkable similarity to autologous fat at the later stages of implantation. And about (18.3 ± 29.3) % of the regenerated adipocytes were still viable after one year. The process of adipogenesis was enhanced by the decrease in inflammatory infiltration and proceeded in parallel with angiogenesis. Statement of significance: The decellularized adipose matrix (DAM) is the only biological scaffold that can spontaneously generate adipocytes in vivo without the need to add exogenous cells. However, in the previous studies, the longest DAM-related animal experiments were about 3 months. The different stages and characteristics of DAM implantation cannot be fully captured. Comprehensive preclinical researches on the initiation, characteristics, and long-term outcomes of DAM-induced adipose tissue regeneration in adulthood is crucial.In this study, we closely observed various aspects of the entire process in vivo from 3 days to 1 year after implantation including fat regeneration, vascular growth, inflammatory reactions as well as matrix degradation and remodeling. The thorough research will contribute to the understanding of stability and dynamic remodeling of DAM regeneration models.

Hydrophilic Components as Key Active Ingredients in Adipose-Derived Matrix Bioscaffolds for Inducing Fat Regeneration.

Decellularized adipose-derived matrix (DAM) has emerged as a promising biomaterial for soft tissue reconstruction. However, due to a lack of research on its complex composition, the understanding of the key components in DAM remains limited, leading to inconsistent adipogenic properties and challenges in optimizing preparation methods purposefully. In this study, it is proposed for the first time that DAM comprises two distinct components: hydrophilic (H-DAM) and lipophilic (L-DAM), each with markedly different effects on fat regeneration. It is confirmed that H-DAM is the key component for inducing fat regeneration due to its enhanced cell-cell and cell-scaffold interactions, primarily mediated by the Hedgehog signaling pathway. In contrast, L-DAM exhibits poor cell adhesion and contains more antigenic components, leading to a higher immunoinflammatory response and reduced adipogenesis. In addition, it is found that intracellular proteins, which are more abundant in H-DAM, can be retained as beneficial components due to their hydrophilicity, contrary to the conventional view that they shall be removed. Accordingly, a purified bioscaffold with unprecedented efficacy is proposed for fat regeneration and reduced immunogenicity. This finding provides insights for developing scaffolds for fat regeneration and promotes the realization of xenotransplantation.

PPAR-γ regulates the polarization of M2 macrophages to improve the microenvironment for autologous fat grafting.

The unpredictable survival rate of autologous fat grafting (AFG) seriously affects its clinical application. Improving the survival rate of AFG has become an unresolved issue in plastic surgery. Peroxisome proliferator-activated receptor-γ (PPAR-γ) regulates the adipogenic differentiation of adipocytes, but the functional mechanism in AFG remains unclear. In this study, we established an animal model of AFG and demonstrated the superior therapeutic effect of PPAR-γ regulation in the process of AFG. From day 3 after fat grafting, the PPAR-γ agonist rosiglitazone group consistently showed better adipose integrity, fewer oil cysts, and fibrosis. Massive macrophage infiltration was observed after 7 days. At the same time, M2 macrophages begin to appear. At day 14, M2 macrophages gradually became the dominant cell population, which suppressed inflammation and promoted revascularization and fat regeneration. In addition, transcriptome sequencing showed that the differentially expressed genes in the Rosiglitazone group were associated with the pathways of adipose regeneration, differentiation, and angiogenesis; these results provide new ideas for clinical treatment.

Dual Network Hydrogels Based on PRP and SA Promote the Retention Rate and Vascularization of Transplanted Fat.

Autologous adipose tissue often experiences ischemia and hypoxia after transplantation, leading to low retention rates and unstable operative impacts due to necrotic absorption. Platelet-rich plasma (PRP) can enhance fat regeneration and increase the fat retention rate after transplantation. However, the quick release of growth factors (GFs) in PRP decreases therapeutic efficiency. This study aimed to achieve a slow release of PRP to promote fat retention. We prepared a dual-network hydrogel (DN gel) based on FDA-approved PRP and sodium alginate (SA) through a simple "one-step" activation process. In vivo study, adipose tissue with saline (control group), SA gel (SA gel group), PRP gel (PRP gel group), and DN gel (DN gel group) was injected subcutaneously into the dorsum of nude mice. At 4 and 12 weeks after injection, tissues were assessed for volume and weight. Hematoxylin and eosin staining (HE) and immunofluorescence staining were performed for histological assessment. DN gel exhibits long-lasting growth factor effects, surpassing conventional clinical PRP gel regarding vascularization potential. In fat transplantation experiments, DN gel demonstrated improved vascularization of transplanted fat and increased retention rates, showing promise for clinical applications. DN gel-assisted lipofilling can significantly improve the retention rate and quality of transplanted fat. DN gel-assisted lipofilling, which is considered convenient, is a promising technique to improve neovascularization and fat survival. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. 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.

The Synergetic Roles of Stromal Vascular Fraction (SVF) and Extracellular Matrix (ECM) on Fat Graft Retention in Nude Mice

Background. Both stromal vascular fraction (SVF) and extracellular matrix (ECM) are of great concern to adipogenesis and angiogenesis. SVF and ECM are rich in adipose tissue and may provide structural and biochemical support and form a microenvironment for free granular fat. The present study was to investigate whether SVF‐gel, a mixture of SVF and ECM harvested by mechanical emulsification, could improve the long‐term volume retention of fat grafts. Methods. Human SVF‐gel of different percentages was mixed with microfat. According to the percentages of SVF‐gel into microfat, 4 groups were included in the study; they are the microfat group, 10% gel group, 30% gel group, and gel group. The fat grafts were transplanted in the subcutaneous layer on each flank in nude mice. The sample volume was measured to evaluate the fat retention rate 90 days post‐transplantation. Tissue integrity, collagen content, numbers of viable adipocytes, and density of blood vessels were examined by further detection. Results. The retention volume rates in the 30% gel group and gel group were significantly higher than the microfat group and the 10% gel group (p < 0.05). Equivalent fat integrity was observed in the four groups. Higher collagen volume, enhanced mRNA expression of VEGF, TNF‐α and adiponectin, more CD31‐positive blood vessels, and more regenerative adipocytes were observed in the 30% gel group and the gel group. The fat tissue in the 30% gel group showed similar structures as the normal fat tissue, while almost of the tissue in the gel group exhibited as fibrous tissue. Conclusion. SVF‐gel could improve fat graft retention while it came to a certain ratio into microfat via proangiogenic effect and fat regeneration which may be provided by SVF and ECM synergistically. SVF‐gel‐assisted fat grafting is a promising strategy to be used in clinical operations.

A novel combined technology for treating hypertrophic scars: adipose tissue extract combined with fractional CO2 laser.

Introduction: Owing to the need for liposuction and its unsuitability for allogeneic transplantation, the clinical application of stromal vascular fraction gel (SVF-gel) combined with fractional CO2 laser for scar treatment is limited. Adipose tissue extract (ATE), rich in cytokines and growth factors, offers a more convenient option for clinical practice as it can be easily prepared using purely physical methods and has low immunogenicity. We aimed to evaluate the effectiveness of ATE combined with fractional CO2 laser in the treatment of hypertrophic scars. Methods: ATE was prepared using discarded liposuction fluid from patients undergoing liposuction. A rabbit ear hypertrophic scar model was established and treated with ATE, fractional CO2 laser, or a combination. PBS was used as a control. The scar appearance and histological changes were observed. The immunohistochemistry method was used to evaluate the expression of α-SMA, while perilipin was detected using immunofluorescence. Additionally, the level of adipogenic signal C/EBPα and PPARγ mRNA was studied. Results: Following treatment, the volume of hypertrophic scar decreased, resulting in a softer texture and thinner dermis. Additionally, there was a decrease in the infiltration of inflammatory cells, and the collagen arrangement became looser and more regular, and the expression of α-SMA also decreased, with the combination of ATE and fractional laser showing the most significant improvement. Moreover, the combination group was found to promote subcutaneous fat regeneration and increase the expression of adipogenic signals C/EBPα and PPARγ. Conclusion: The combination of ATE and fractional CO2 laser treatment has been shown to inhibit the development of hypertrophic scars. This effect may be attributed to the enhancement of adipogenesis and decrease in collagen deposition.

Effect of radiation sterilization on the ability to induce adipose regeneration in vivo in decellularized adipose-derived matrix.

Decellularized adipose-derived matrix (DAM), a biological scaffold that can induce adipose regeneration. The balance between its sterilization efficiency and its ability to maintain in situ adipose regeneration should be considered in terminal sterilization. The purpose of this study was to investigate the effects of radiation sterilization of cobalt-60 (60 Co)with different doses on adipogenesis induced by different forms of DAM, so as to reduce radiation dose under the premise of safe and effective sterilization and ensure adipogenesis induced by DAM in vivo. High dose (25kGy) and low dose (5kGy) radiation were used to sterilize freeze-dried and wet DAM, respectively. The sterilization efficiency, macro and micro characteristics, mechanical and mechanical properties of DAM were compared, and then implanted into the immunocompromised mice to evaluate the adipose regeneration. Under the two radiation doses, no microbial growth was found in the freeze-dried and wet DAM sterility tests, and no significant changes were observed in the macro and micro structures. In terms of mechanical properties, the elastic modulus of high dose freeze-dried DAM decreased significantly (p<0.001). In vivo animal experiments, the freeze-dried DAM irradiated with high dose almost completely lost its function of adipogenesis in vivo. Although the wet DAM irradiated with high dose could induce fat regeneration in the early stage, the adipocyte deformation and atrophy appeared in the later stage. The freeze-dried and wet DAM after low dose irradiation was similar to the wet DAM without irradiation in the blank control, which could maintain excellent adipogenic and angiogenic functions in vivo. High dose 60 Co irradiation can completely destroy the ability of freeze-dried DAM to induce adipose regeneration in situ, while low dose irradiation (5kGy) can effectively sterilize the DAM without damaging in vivo induced adipose regeneration. Radiation has more damage to freeze-dried DAM than wet DAM in adipogenesis properties.

Ultra-condensed Fat: A Novel Fat Product for Volume Augmentation.

Fat transplantation retention rate is individualized and unpredictable. The presence of blood components and oil droplets in the injected lipoaspirate increases inflammation and fibrosis in a dose-dependent manner, and is probably the key factor associated with poor retention. This study describes a volumetric fat grafting strategy based on optimization of grafts via screening intact fat particles and absorbing free oil droplets and impurities. Centrifuged fat components were analyzed by n-hexane leaching. A special device was applied to de-oil intact fat components and obtain ultra-condensed fat (UCF). UCF was evaluated by scanning electron microscopy, particle size analysis, and flow cytometric analysis. Histological and immunohistochemical changes were investigated in a nude mouse fat graft model over 90days. The lower 50% of centrifuged fat was concentrated to 40% of the original volume to obtain UCF. In UCF, the free oil droplet content was less than 10%, more than 80% of particles were larger than 1000µm, and architecturally important fat components were present. The retention rate of UCF was significantly higher than that of Coleman fat on day 90 (57.5 ± 2.7% vs. 32.8 ± 2.5%, p < 0.001). Histological analysis detected small preadipocytes with multiple intracellular lipid droplets on day 3 in UCF grafts, indicative of early adipogenesis. Angiogenesis and macrophage infiltration were observed in UCF grafts soon after transplantation. Adipose regeneration with UCF involves rapid macrophage infiltration and exit, resulting in angiogenesis and adipogenesis. UCF may serve as a lipofiller which is beneficial for fat regeneration. 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 http://www.springer.com/00266 .

Body-Shaping Membrane to Regenerate Breast Fat by Elastic Structural Holding

Tissue regeneration requires structural holding and movement support using tissue-type-specific aids such as bone casts, skin bandages, and joint protectors. Currently, an unmet need exists in aiding breast fat regeneration as the breast moves following continuous body motion by exposing the breast fat to dynamic stresses. Here, the concept of elastic structural holding is applied to develop a shape-fitting moldable membrane for breast fat regeneration ("adipoconductive") after surgical defects are made. The membrane has the following key characteristics: (a) It contains a panel of honeycomb structures, thereby efficiently handling motion stress through the entire membrane; (b) a strut is added into each honeycomb in a direction perpendicular to gravity, thereby suppressing the deformation and stress concentration upon lying and standing; and (c) thermo-responsive moldable elastomers are used to support structural holding by suppressing large deviations of movement that occur sporadically. The elastomer became moldable upon a temperature shift above Tm. The structure can then be fixed as the temperature decreases. As a result, the membrane promotes adipogenesis by activating mechanotransduction in a fat miniature model with pre-adipocyte spheroids under continuous shaking invitro and in a subcutaneous implant placed on the motion-prone back areas of rodents invivo.

Review on the Role of Fructose in Non-Alcoholic Fatty Liver Diseases

Non-alcoholic fatty liver disease (NAFLD) is a clinical liver condition characterized by accumulation of fat in the liver that can develop into non-alcoholic steatohepatitis (NASH) without any evidence of excessive alcohol consumption. NASH involves liver inflammation, necrosis, fibrosis, cirrhosis, and eventually liver cancer. Excessive intake of fructose is known to promote non-alcoholic fatty liver (NAFLD), because this sugar is both a substrate and an inducer of liver fat regeneration. In addition to the hepatic lipogenic effects, the consumption of fructose can also cause liver inflammation and cellular stress, such as oxidative and endoplasmic stress, which are conducive to the progression of steatosis to non-alcoholic steatohepatitis (NASH). Fructose also has direct and indirect effects on peripheral levels. For example, excessive fructose intake is associated with changes in the gut micro biota, which can lead to a worsening of the disease. Currently, there is no known treatment for NAFLD. Therefore, treatment is based on lifestyle changes, including changes in diet and physical exercise.

Characterized the Adipogenic Capacity of Adipose-Derived Stem Cell, Extracellular Matrix, and Microenvironment With Fat Components Grafting.

Background: Autologous fat grafting has been a widely used technique; however, the role of adipose-derived stem cells (ASCs), extracellular matrix (ECM), and microenvironment in fat regeneration are not fully understood.Methods: Lipoaspirates were obtained and processed by inter-syringe shifting to remove adipocytes, yielding an adipocyte-free fat (Aff). Aff was then exposed to lethal dose of radiation to obtain decellularized fat (Df). To further remove microenvironment, Df was rinsed with phosphate-buffered saline (PBS) yielding rinsed decellularized fat (Rdf). Green fluorescent protein (GFP) lentivirus (LV-GFP)-transfected ASCs were added to Df to generate cell-recombinant decellularized fat (Crdf). Grafts were transplanted subcutaneously into nude mice and harvested over 3 months.Results: Removal of adipocytes (Aff) didn’t compromise the retention of fat grafts, while additional removal of stromal vascular fraction (SVF) cells (Df) and microenvironment (Rdf) resulted in poor retention by day 90 (Aff, 82 ± 7.1% vs. Df, 28 ± 6.3%; p < 0.05; vs. Rdf, 5 ± 1.2%; p < 0.05). Addition of ASCs to Df (Crdf) partially restored its regenerative potential. Aff and Crdf exhibited rapid angiogenesis and M2-polarized macrophages infiltration, in contrast to impaired angiogenesis and M1-polarized inflammatory pattern in Df. GFP + ASCs participated in angiogenesis and displayed a phenotype of endothelial cells in Crdf.Conclusion: Adipose ECM and microenvironment have the capacity to stimulate early adipogenesis while ECM alone cannot induce adipogenesis in vivo. By directly differentiating into endothelial cells and regulating macrophage polarization, ASCs coordinate early adipogenesis with angiogenesis and tissue remodeling, leading to better long-term retention and greater tissue integrity.

Cellular basis of omentum activation and expansion revealed by single-cell RNA sequencing using a parabiosis model

Although the physiological function of the omentum remains elusive, it has been proposed that it plays an important role in fat storage, immune regulation, and regeneration of injured tissues and organs. Although the omentum undergoes expansion upon activation, reports on the accurate assessment of increased cell types and the origin of the increased cells remain limited. To investigate this aspect, the omenta of parabiotic mice were subjected to activation using distinct fluorescent markers and single-cell RNA sequencing (scRNA-seq) was performed to identify circulation-derived omental cells. We found that a considerable number of circulating cells contributed to the activation of the omentum. The omental cells derived from circulating cells exhibited morphological features similar to those of fibroblasts. scRNA-seq revealed the existence of a novel cell population that co-expressed macrophage and fibroblast markers in the activated omentum, suggesting that it corresponded to circulating macrophage-derived fibroblast-like cells. Lineage tracing experiments revealed that most fibroblasts in the activated omentum were not derived from WT1-positive mesenchymal progenitors. The cell cluster also expressed various chemokine genes, indicating its role in the activation and recruitment of immune cells. These results provide important information regarding the activation of the omentum.

Decellularized adipose tissue: A key factor in promoting fat regeneration by recruiting and inducing mesenchymal stem cells

BackgroundDecellularized adipose tissue (DAT) has attracted much attention due to its wide range of sources and adipose regeneration capacity. However, the lipogenic efficiency of DAT is still controversial due to its unclear mechanism. To this point, it is crucial to clarify the mechanism of DAT in promoting adipose regeneration Objective: This study aims to explore the mechanism of DAT promoting adipose regeneration and survival mechanism of DAT transplantation in vivo. MethodsDAT preparation by repeated freeze-thaw, enzymatic digestion, and isopropanol degreasing. Histology, DAPI, immunohistochemistry, immunofluorescence and scanning electron microscopy confirmed the efficacy and reproducibility of these approaches. BM-MSCs, ADSCs and UCMSCs were cocultured with DAT for 14 days and then stained with oil red O. Adipogenic genes of three MSCs were detected by RT-PCR. DAT and adipose tissue were transplanted subcutaneously into the back of nude mice to observe medium and long-term morphological changes, vascularization, and lipid-forming efficiency. Mass spectrometry (MS)-based proteomic to analyze the adipogenic protein contents of DAT and adipose tissue. ResultsThe DAT without any cellular components but with an abundance of collagen; neither DNA nor lipids were detected. Seeding experiments with MSCs indicated that the DAT provided an inductive microenvironment for adipogenesis, supporting the expression of the master regulators PPARγ. Within four months after transplantation, HE morphology of DAT was identical to adipose cells. Immunofluorescence markers CD31 and perilipin were increased in DAT, while the retention rate gradually decreased over time, eventually accounting for 33.7% of the original volume. MS-based proteomic analyses identified 1013 types of proteins in adipose tissue and 29 proteins in the DAT. Analyses of GO and KEGG databases suggested that DAT contained a variety of proteins involved in fat metabolism. ConclusionsDAT can interact with different types of MSCs and ultimately achieve adipose regeneration. The presence of multiple adipogenic proteins in DAT make it play a vital role in adipose regeneration. DAT is expected to be an ideal bio-derived scaffold for adipose tissue engineering.

The Application and Mechanism of Action of External Volume Expansion in Soft Tissue Regeneration.

The reconstruction of large-volume soft tissue defects is a major problem in plastic surgery. Many plastic surgeons have focused on external volume expansion (EVE) because of its capacity to promote regeneration of soft tissues, including breast, subcutaneous fat, and skin. EVE is a minimally invasive and less costly tissue engineering approach that has shown great clinical potential. However, many challenges still need to be addressed before such technology can become a common clinical practice. Basic in vivo and in vitro studies have been performed to determine the possible mechanisms by which EVE promotes tissue regeneration and to design optimized animal models. EVE application was found to facilitate cell proliferation and migration, enhance adipogenesis, improve angiogenesis, and provide available space for soft tissue growth. Understanding the mechanical and chemical signals associated with EVE during tissue regeneration may enable the clinical adaptation of this technology. This article reviews the clinical application of EVE techniques, describes preclinical animal models, and evaluates the possible mechanisms by which EVE induces tissue regeneration. Impact statement The reconstruction of large-volume soft tissue defects is a major problem in plastic surgery. External volume expansion (EVE) is a minimally invasive and less costly tissue engineering approach that has shown promise in clinical applications. This article reviews the clinical application of EVE techniques, describes preclinical animal models, and evaluates the possible mechanisms by which EVE induces tissue regeneration.

Delayed two steps PRP injection strategy for the improvement of fat graft survival with superior angiogenesis

Platelet-rich plasma (PRP) has been widely used to improve the fat retention rate in autologous fat transplantation since it possesses a good angiogenesis capability in vivo. However, due to the short half-life of growth factors released from PRP and its uneven distribution in injected fat tissue, the strategy of PRP in fat transplantation needs further improvement. Since the capillaries started to grow into fat grafts in 1 week and vascular growth peaks in the second week after transplantation, we hypothesized that delayed two-steps PRP injection into the interior of grafts, accompanied with the extent of neovascularization might theoretically promote microvessel growth inside transplanted adipose tissue. 24 nude mice were divided into three groups: Blank group (0.35 mL fat mixed with 0.15 mL saline, N = 8), Single step group (0.35 mL fat mixed with 0.15 mLPRP, N = 8), and Two steps group (0.35 mL fat (day 0) + 0.075 mL PRP (day 7) + 0.075 mL PRP (day 14), N = 8). At 6 and 14 weeks post-transplantation, grafts were dissected, weighted, and assessed for histology, angiogenesis, fat regeneration and inflammation level. The weight and volume of the fat samples revealed no statistical difference among the three groups at 6 weeks after fat transplantation. The weight and volume of the Two steps group fat samples showed significantly higher compared to that in Blank and Single step groups at 14 weeks after fat transplantation (weight: 137.25 ± 5.60 mg versus 87.5 ± 3.90 mg,106.75 ± 2.94 mg, respectively; volume: 0.13 ± 0.01 mL versus 0.08 ± 0.01 mL, 0.09 ± 0.01 mL, respectively). Histological assessments indicated that delayed two-steps PRP injection strategy helps to improve adipose tissue content and reduce the composition of fibrous connective tissue at 14 weeks after fat transplantation. At 6 weeks and 14 weeks after transplantation, CD31 immunofluorescence indicated that delayed two-steps PRP injection strategy helps to improve angiogenesis and significantly higher compared to that in Blank and Single step groups (6 weeks: 28.75 ± 4.54 versus 10.50 ± 2.06, 21.75 ± 1.85; 14 weeks: 21.75 ± 2.86 versus 9.87 ± 2.08, 11.75 ± 1.47, respectively). Preadipocyte count indicated delayed two-steps PRP injection strategy might promote fat regeneration and significantly higher compared to that in Blank and Single step groups at 14 weeks (129.75 ± 6.57 versus 13.50 ± 3.50, 17.12 ± 6.23, respectively). In this study, we demonstrated that the novel delayed two-steps PRP injection strategy remarkably enhanced the long-term fat retention rate and improved the neovascularization extent in the interior of the fat graft. Platelet-rich plasma, Delayed two-steps injection, Angiogenesis, Fat transplantation

Introduction of ligated vessels promote the retention and regeneration of free fat: constructing a fat flap in tissue engineering chamber

ABSTRACT Background: Breast reconstruction with fat grafting has an unstable retention rate due to insufficient revascularization. Tissue Engineering Chamber (TEC) model can promote tissue regeneration in the chamber by introducing ligated vessels around the tissue. We introduced ligated vessels with free fat graft to investigate the retention rate and revascularization of grafted fat that in TEC model. Methods: SD rats (n=24) was divided into 3 groups randomly. Group A: Standard TEC model was constructed; Group B: the epigastric vessel bundles were dissected from the fat flap and ligated, fat flap was cut into granules and planted into the chamber; Group C: Free fat was planted in the chamber. At week 6, samples in the chamber were harvested. Results: Significant volume increase was observed in group A and B, while the volume decreased in group C (P<0.05). Regeneration morphology could be found according to the histological observation in A and B. Micro CT results showed the ligated vessels into grafted fat sprouting robustly, coordinated with volume changes. Conclusion: Fat grafts in TEC model could not only survive but also regenerate. The combination of fat graft and TEC could fabricate a vascularized fat flap, which was a promising method in breast reconstruction. Abbreviations: VOI: Volumes of Interest; TEC: Tissue Engineering Chamber; CAL: Cell Assisted Lipotransfer.

Osteogenic and chondrogenic differentiation effect of neohesperidin dihydrochalcones in human adipose‐derived stem cells

Reactive oxygen species (ROS) are highly reactive which cause damage to various cellular functions such as proliferation, differentiation, and survival. Nuclear factor erythroid 2‐related factor‐2 (Nrf2) promotes expression of antioxidant enzyme such as heme oxygenase 1 (HO‐1) and NAD(P)H:Quinone Oxidoreductase 1 (NQO‐1). Neohesperidin is a compound originated from peel of citrus fruits and its hydrogenated form, neohesperidin dihydrochalcone (NHDC) has antioxidant activity. Human adipose‐derived stem cells (hADSC) are multipotent adult stem cells responsible for the regeneration of fat, bone and cartilage. Thus, we observed the antioxidant effects of NHDC derivatives on hADSC through Nrf2 activation. Upregulation of ROS inhibits osteogenic differentiation of hADSCs, in part through inhibition of signaling pathway, which is essential for bone development and maintenance. ROS levels regulate inhibition of proliferation and modulate initiation of the hypertrophic changes in chondrocytes. Cellular viability and compounds' proper concentrations were set by MTS assay. The effects of these compounds on suppressing oxidative stress and hADSCs differentiation into osteocytes and chondrocytes was investigated using Alizarin Red staining, Alcian Blue staining, reverse‐transcriptase polymerase chain reaction (RT‐PCR), and Western blotting. The results demonstrate that NHDC derivatives play an efficacious role in inducing osteogenic and chondrogenic differentiation of hADSCs through reducing cellular oxidative stress.Support or Funding InformationThis research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education 2018R1D1A1B07049077This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Paradoxical effects of the epigenetic modifiers 5-aza-deoxycytidine and suberoylanilide hydroxamic acid on adipogenesis

Adipogenesis is an important biological process that is linked to obesity and metabolic disorders. On the other hand, fat regeneration is crucial as a restorative approach following mastectomy or severe burn injury. Furthermore, optimizing an in-vitro model of adipogenesis, which would help in understanding the possible effects and/or side effects of fat-soluble drugs and anti-obesity remedies, in addition to the developmental studies. Epigenetic is an important factor that is involved in cellular differentiation and commitment. This study aimed at investigating the effect of DNA methylation and histone deactylases inhibitors, 5-Aza-deoxycytidine (5-Aza-dC) and Suberoylanilide hydroxamic acid (SAHA), on the adipogenic differentiation process. The two modifiers were applied according to our previously published protocol, followed by three cycles of a classical, two-step adipogenesis protocol. The cells pretreated with SAHA showed enhanced expression of the many adipogenic genes, including peroxisome proliferator-activated receptor-γ as well as the accumulation of intracytoplasmic fat as shown by oil red and Nile red staining and the secretion of adipokines, such as MCP-1 and IP-10. On contrary, 5-Aza-dC inhibited all these markers. In conclusion, adding the reported step with SAHA to the differentiation protocols could have an impact on the progress of the in-vitro fat regenerative approach. The possible role of 5-Aza-dC in the inhibition of adipogenesis can be of clinical interest and will need further characterization in the future.

Fate of Free Fat Grafts with or without Adipogenic Adjuncts to Enhance Graft Outcomes.

Free fat grafting is popular, but it is still unclear how it works. Although focusing on graft survival seems an obvious direction for improving clinical results, the authors' research suggests that long-term volume retention is in part attributable to new fat regeneration. Measures to facilitate adipogenesis may therefore be equally important. To investigate the relative roles of survival and regeneration of fat grafts, the authors measured the fate of human lipoaspirate implanted into the scalps of immunodeficient mice, with and without stromal vascular fraction and a porcine extracellular matrix (Adipogel). Specifically, the authors were interested in volume retention, and the composition of implanted or regenerated tissue at 6 and 12 weeks. Free fat grafts exhibited poor volume retention and survival. Almost all of the injected human adipocytes died, but new mouse fat formed peripheral to the encapsulated fat graft. Adipogel and stromal vascular fraction improved proliferation of murine fat and human vasculature. Human CD34 stromal cells were present but only in the periphery, and there was no evidence that these cells differentiated into adipocytes. In the authors' model, most of the implanted tissue died, but unresorbed dead fat accounted substantially for the long-term, reduced volume. A layer of host-derived, regenerated adipose tissue was present at the periphery. This regeneration may be driven by the presence of dying fat, and it was enhanced by addition of the authors' adipogenic adjuncts. Future research should perhaps focus not only on improving graft survival but also on enhancing the adipogenic environment conducive to fat regeneration.

Effects of malvidin, cyanidin and delphinidin on human adipose mesenchymal stem cell differentiation into adipocytes, chondrocytes and osteocytes

BackgroundAnthocyanidins are plant phytochemicals found at high concentrations in berries, vegetables and flowers. Anthocyanidins have been extensively investigated due to their antioxidative, antidiabetic and anti-inflammatory effects. Few studies show that anthocyanidins decrease obesity and improve bone density. However, the effects of anthocyanidins on tissue regeneration have not been sufficiently clarified. Human mesenchymal stem cells (MSCs) are multipotent adult stem cells responsible for the regeneration of fat, bone and cartilage. Although MSCs are often used for screening of biologically active compounds, so far, the effect of anthocyanidins on MSC differentiation has not been addressed. PurposeThe aim of this study was to analyse the effect of anthocyanidins malvidin, cyanidin and delphinidin on adipose tissue-derived MSC differentiation into adipocytes, osteocytes and chondrocytes. Study design and methodsDifferentiation into adipocytes, osteocytes and chondrocytes was carried out in the defined cell culture conditions in the presence or absence of malvidin, cyanidin and delphinidin. The differentiation was confirmed by cytochemical staining and tissue-specific gene and protein expression. Antiobesity and anti-diabetes drug liraglutide was used as a reference drug in this study. ResultsDelphinidin inhibited MSC adipogenesis and downregulated FABP4 and adiponectin genes. Malvidin induced a significantly higher accumulation of calcium deposits in MSCs comparing to untreated MSCs, as well as upregulated the osteocyte-specific gene BMP-2 and Runx-2 expression and induced BMP-2 secretion. Cyanidin and delphinidin demonstrated a chondrogenesis stimulating effect by upregulation of Col2a1 and aggrecan. ConclusionAltogether, our data show that anthocyanidins malvidin, cyanidin and delphinidin exert favourable effects on MSC osteogenesis and chondrogenesis whereas delphinidin inhibits adipogenesis. These results suggest that anthocyanidin effects on tissue regeneration could be further analysed in depth in vivo.