Adipokines-enriched adipose extract restores skin barrier and ameliorates inflammatory dysregulation in an atopic dermatitis mouse model.

BACKGROUND: Atopic dermatitis (AD) is a chronic dermatosis with high incidence worldwide characterized by skin barrier abnormalities and immune dysregulation. Conventional therapies are usually limited by side effects and high cost. Given the anti-inflammatory and repairing properties, adipokines are increasingly considered as promising therapeutic agents for dermatoses. Adipose collagen fragments (ACF), a novel adipokine-enriched product, may alleviate AD through modulating immune microenvironment and restoring skin barrier. METHODS: ACF was extracted from adipose tissue via high-speed homogenization (10000 rpm/min, 1min) and centrifugation (3000 g, 3min). Ovalbumin-induced AD female BALB/c mice (6-week-old) were intradermally injected with 0.2ml of ACF or PBS (negative control), with normal mice being set as normal control (n=6). Dermatitis severity, inflammatory metrics (epidermal thickness, infiltrated mast cells, Th-type cytokines expression), and skin barrier-related metrics (transepidermal water loss [TEWL], skin barrier-related proteins expression) were evaluated after the AD induction period (day 50). ACF-derived bioactive components were also evaluated using proteomic analysis. RESULTS: ACF-derived adipokines contained anti-inflammatory, skin barrier- and lipid biosynthesis-related components. ACF treatment decreased dermatitis severity (6.2±1.8, p<0.0001), epidermal thickness (25.7±12.8 µm, p=0.0045), infiltrated mast cells (31.3±12.4 cells/field, p=0.0475), and Th-type cytokines expression (INF-γ, TNF-α, IL-4, IL-4R, IL-13, and IL-17A; p<0.05) in AD skins. TEWL (29.8±13.8 g/m 2.h, p=0.0306) and skin barrier-related proteins expression (filaggrin: 14258±4375, p=0.0162; loricrin: 6037±1728, p=0.0010; claudin-1: 20043±6406, p=0.0420; ZO-1: 4494±1114, p=0.0134) were also improved. CONCLUSIONS: ACF improved AD in murine model by ameliorating inflammatory dysregulation and skin barrier defects (Graphical abstract, Supplementary Digital Content 1). Further validation is needed in more advanced animal models.

Skin-associated adipocytes in skin barrier immunity: A mini-review.

The skin contributes critically to health via its role as a barrier tissue against a multitude of external pathogens. The barrier function of the skin largely depends on the uppermost epidermal layer which is reinforced by skin barrier immunity. The integrity and effectiveness of skin barrier immunity strongly depends on the close interplay and communication between immune cells and the skin environment. Skin-associated adipocytes have been recognized to play a significant role in modulating skin immune responses and infection by secreting cytokines, adipokines, and antimicrobial peptides. This review summarizes the recent understanding of the interactions between skin-associated adipocytes and other skin cells in maintaining the integrity and effectiveness of skin barrier immunity.

Adipose-derived stem cells regulate CD4+ T-cell-mediated macrophage polarization and fibrosis in fat grafting in a mouse model.

Autologous fat grafting is becoming increasingly common worldly. However, the long-term retention of fat grafting is still unpredictable due to the inevitable fibrosis arising during tissue repair. Fibrosis may be regulated by T-cell immune responses that are influenced by adipose-derived stem cells (ASCs). Therefore, we hypothesized that overly abundant ASCs might promote fibrosis by promoting T-cell immune responses to adipose tissue. We performed 0.3 ml fat grafts with 104/ml, 106/ml and 108/ml ASCs and control group in C57 BL/6 mice in vivo. We observed retention, fibrosis, T-cell immunity, and macrophage infiltration over 12 weeks. Besides, CD4+ T-helper 1 (Th1) cells and T-helper 2 (Th2) cells were co-cultured with ASCs or ASCs conditioned media (ASCs-CM) in vitro. We detected the ratio of Th2%/Th1%. Results showed that the retention rate was higher in 104 group, while even lower in 108 group with significantly increased inflammation and fibrosis than control group at week 12 in vivo. There was no significance between control group and 106 group. Also, 108 group increased the infiltration of M2 macrophages, CD4+ T-cells and Th2/Th1 ratio. In vitro, the ratio of Th2%/Th1% induced by ASCs-transwell group was higher than ASCs-CM group and showed concentration-dependent. Accordingly, high concentrations of ASCs in adipose tissue can promote Th1-Th2 shifting, and excessive Th2 cells might promote the persistence of M2 macrophages and increase the level of fibrosis which lead to a decrease in the long-term retention of fat grafts. Also, we found ASCs promoted Th1-Th2 shifting in vitro.

Density-Based High-Quality Fat: Characterization and Correlation with Different Body Fat Ratio.

BACKGROUND: Lipoaspirate can be divided into high-quality fat and low-quality fat using Coleman's centrifugation by adding 0.935 g/ml marker float; the ratio obtained by different individuals is different. OBJECTIVES: This study aimed to examine the HQF obtained from different individuals and establish the relationship between individual body data and HQF. METHODS: We used Coleman's centrifugation method (1200 g, 3 min) with 0.935 g/ml density float to process lipoaspirate and collect HQF from different individuals for the analysis of fat characteristics and in vivo grafting. RESULTS: The HQF obtained from different individuals had similar stromal vascular fraction cell numbers and extracellular matrix content. In animal experiments at different time points (especially 12 weeks), the appearance, retention rate, hematoxylin and eosin staining, and immunohistochemistry results of HQF grafts were similar, while being different from those of Coleman fat. The HQF obtained from individuals with higher body fat ratio was less than those with lower body fat ratio. Following the establishment of the relationship between high-quality fat percentage and the body fat ratio of the donors, we proposed an innovative calculation formula model for the required lipoaspirate. CONCLUSIONS: HQF obtained from different individuals has similar fat characteristics, transplantation process, and outcome. The HQF percentage obtained from different individuals is negatively correlated with the body fat ratio. The amount of liposuction can be predicted using the proposed formula and improve the predictability of fat transplantation. LEVEL OF EVIDENCE IV: 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.

Adipose-derived stem cells ameliorate atopic dermatitis by suppressing the IL-17 expression of Th17 cells in an ovalbumin-induced mouse model.

BACKGROUND: Mesenchymal stem cells (MSCs) have therapeutic potential for atopic dermatitis (AD) owing to their immunoregulatory effects. However, the underlying mechanisms associated with the therapeutic efficacy of MSCs on AD are diverse and related to both cell type and delivery method. OBJECTIVES: This study investigated the therapeutic effect and mechanisms of adipose-derived stem cells (ADSCs) on AD using an ovalbumin (OVA)-induced AD mouse model. METHODS: AD mice were subcutaneously injected with mouse ADSCs, cortisone, or PBS, and the therapeutic effects were determined by gross and histological examinations and serum IgE levels. Additionally, qPCR, RNA-sequencing analyses of skin samples and co-culture of ADSCs and Th17 cells were conducted to explore the underlying therapeutic mechanisms. RESULTS: ADSCs treatment attenuated the AD pathology, decreased the serum IgE levels, and decreased mast cells infiltration in the skin of the model mice. Moreover, tissue levels of IL-4R and Th17-relevant products (IL-17A, CCL20, and MMP12) were suppressed in the ADSC- and cortisone-treated groups. Genomics and bioinformatics analyses demonstrated significant enrichment of inflammation-related pathways in the downregulated genes of the ADSC- and cortisone-treated groups, specifically the IL-17 signaling pathway. Co-culture experiments revealed that ADSCs significantly suppressed the proliferation of Th17 cells and the expression of proinflammatory cytokines (IL-17A and RORγT). Furthermore, expression levels of PD-L1, TGF-ß, and PGE2 were significantly upregulated in co-cultured ADSCs relative to those in monocultured ADSCs. CONCLUSION: ADSCs ameliorate OVA-induced AD in mice mainly by downregulating IL-17 secretion of Th17 cells.

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.

The effects of macrophage-mediated inflammatory response to the donor site on long-term retention of a fat graft in the recipient site in a mice model.

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.

Identification of High-Quality Fat Based on Precision Centrifugation in Lipoaspirates Using Marker Floats.

BACKGROUND: Centrifugation creates "graded densities" of fat with varying cellular and biological compositions that influence graft retention. This study aimed to find an accurate method to identify fat fractions that are suitable for implantation. METHODS: Five marker floats (0.925, 0.930, 0.935, 0.940, and 0.945 g/ml) were added to human lipoaspirates that were then centrifuged at 1200 g for 3 minutes to grade the density of centrifuged lipoaspirates. After centrifugation, four fat fractions divided by floats were collected for fat characteristics analysis and in vivo grafting, with Coleman fat as a control. RESULTS: Fat characteristics varied significantly between the centrifuged fat fractions divided by the 0.935-g/ml marker float. Compared with low-quality fat (<0.935 g/ml), high-quality fat (>0.935 g/ml) contains more stromal vascular fraction, adipose-derived stem cells, and extracellular matrix content. Furthermore, adipocytes were found to be significantly smaller in high-quality fat than in low-quality fat, and high-quality fat persisted at a greater volume compared with low-quality fat in vivo at week 12. CONCLUSIONS: High-quality fat contains more stromal vascular fraction cells, extracellular matrix content, and small adipocytes, leading to the highest implant volume retention, whereas low-quality fat contains more fragile large adipocytes, leading to the least volume retention. Marker floats can be used to grade the density of lipoaspirates, with fat greater than 0.935 g/ml recommended as a suitable alternative for implantation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Optimized adipose tissue engineering strategy based on a neo-mechanical processing method.

Decellularized adipose tissue (DAT) represents a promising scaffold for adipose tissue engineering. However, the unique and prolonged lipid removal process required for adipose tissue can damage extracellular matrix (ECM) constituents. Moreover, inadequate vascularization limits the recellularization of DAT in vivo. We proposed a neo-mechanical protocol for rapidly breaking adipocytes and removing lipid content from adipose tissue. The lipid-depleted adipose tissue was then subjected to a fast and mild decellularization to fabricate high-quality DAT (M-DAT). Adipose liquid extract (ALE) derived from this mechanical process was collected and incorporated into M-DAT to further optimize in vivo recellularization. Ordinary DAT was fabricated and served as a control. This developed strategy was evaluated based on decellularization efficiency, ECM quality, and recellularization efficiency. Angiogenic factor components and angiogenic potential of ALE were evaluated in vivo and in vitro. M-DAT achieved the same decellularization efficiency, but exhibited better retention of ECM components and recellularization, compared with those with ordinary DAT. Protein quantification revealed considerable levels of angiogenic factors (basic fibroblast growth factor, epidermal growth factor, transforming growth factor-ß1, and vascular endothelial growth factor) in ALE. ALE promoted tube formation in vitro and induced intense angiogenesis in M-DAT in vivo; furthermore, higher expression of the adipogenic factor PPARγ and greater numbers of adipocytes were evident following ALE treatment, compared with those in the M-DAT group. Mechanical processing of adipose tissue led to the production of high-quality M-DAT and angiogenic factor-enriched ALE. The combination of ALE and M-DAT could be a promising strategy for engineered adipose tissue construction.