Extracellular vesicles (EVs) derived from human adipose-derived stem cells (hADSCs) possess the proangiogenic potential for ischaemic diseases. Thus, our study aimed to evaluate the therapeutic effects of hADSC-EVs on fat grafting and explore the mechanism of hADSC-EVs promoting angiogenesis. The EVs released by hADSCs incubated under normal or hypoxic conditions were employed to supplement fat grafting in a nude mouse model. The proliferation, migration, tube formation and vascular endothelial growth factor (VEGF) secretion of vascular endothelial cells cocultured with two kinds of hADSC-EVs were analysed. MicroRNA sequencing was performed to reveal the species and content of microRNAs in hADSC-EVs, the key microRNAs were blocked, and their effect in promoting angiogenesis was detected via above protocols as a reverse proof. The results demonstrate that hADSC-EVs could improve the survival of fat grafts by promoting exogenous angiogenesis and enhance the proliferation, migration, tube formation and VEGF secretion of vascular endothelial cells. In addition, the pro-angiogenic effect of hADSC-EVs in vivo and vitro could be enhanced by hypoxic pretreatment. We found that the let-7 family, a kind of hypoxic-related microRNA, is enriched in hypoxic hADSC-EVs that contribute to angiogenesis via the let-7/argonaute 1 (AGO1)/VEGF signalling pathway.Fat grafting has been widely used for soft tissue filling in cosmetic plastic surgery (such as breast augmentation and facial rejuvenation) and reconstruction surgery due to several diseases (such as soft tissue defects caused by facial muscle atrophy, tumour excision and breast reconstruction after breast cancer surgery) 1 . The technology of fat grafting has made significant advances in the past decades, being considered less technically challenging because of the sophisticated liposuction technique, low donor site morbidity after transplantation and reduced host immune responses 1-3 . However, the main limitation is the necrosis and absorption of grafts due to poor revascularization 4,5 . Therefore, the survival of fat grafts can still be improved.The process of liposuction and injection leads to devascularisation and ischaemic injury of the adipose tissue. Since the adipose tissue is less tolerant to ischaemia 3 , early revascularization of the transplanted tissue is critical for the survival of fat grafts 4 . Various studies have demonstrated the promotion of angiogenesis by mesenchymal stem/stromal cells (MSCs). Yoshimura et al. 6 developed a cell-assisted lipotransfer (CAL) strategy to co-transplant autologous adipose tissue with adipose stromal cells. The meta-analysis performed by Laloze et al. 7 demonstrated that the CAL technology can significantly improve survival from 44 to 64%. With the progress on the research of the biological functions of MSCs, for fat grafting, the paracrine effect of adipose-derived stem cells (ADSCs) play important roles in angiogenesis and tissue regeneration 8 . In addition, studies in recent years have www.nature.com/scientificreports...

Background Preventing scar formation during wound healing has important clinical implications. Numerous studies have indicated that adipose-derived stem cell culture mediums, which are rich in cytokines and extracellular vesicles (EVs), regulate matrix remodeling and prevent scar formation after wound healing. Therefore, using a rabbit scar model, we tried to demonstrate which factor in adipose-derived stem cell culture mediums plays a major role in preventing scar formation (EVs or cytokines), as well as revealing the underlying mechanism. Methods Human adipose-derived stem cells (hASCs) were isolated from the subcutaneous adipose tissue of a healthy female donor. The surface CD markers of third-passage hASCs were analyzed by flow cytometry. The adipogenic differentiation capacity of the hASCs was detected using Oil O staining. A cultured medium of third- to five-passage hASCs was collected for EV and EV-free medium isolations. Extracellular vesicles were characterized using transmission electron microscopy, NanoSight, and the Western blotting for surface markers CD63, TSG101, and Alix. The EV-free medium was characterized by Western blotting for vascular endothelial growth factor A (VEGFA), platelet derived growth factor B (PDGFB), and transforming growth factor β 1 (TGFβ1). Eight-millimeter-diameter wounds were created on the ventral side of both ears of 16 New Zealand rabbits. A total of 0.1 mL of the human adipose-derived stem cell–extracellular vesicle (hASC-EV) or EV-free medium was locally injected into wounds made on the right ears during wound healing. Meanwhile, equal amounts of phosphate buffer saline were injected into the left ears as a control. Biopsies of the wounded skin and surrounding tissue were excised on postoperative day 28 and subjected to hematoxylin and eosin (H&E), Masson, and α-SMA immunofluorescence staining. The protein expression of α-SMA and collagen I in both scar tissues and the normal skin were evaluated via Western blotting. Results The hASCs expressed high levels of 49d, CD90, CD105, and CD73 but did not express CD34 or CD45. The hASCs differentiated into adipocytes under an adipogenic induction medium. Under transmission electron microscopy, the hASC-EVs were circular, bilayer membrane vesicles and approximately 95% of the particles were between 50 and 200 nm in size. The hASC-EVs expressed the same surface markers as EVs, including CD63, TSG101, and Alix and displayed little expression of VEGFA, PDGFB, and TGFβ1. The EV-free medium had a high expression of VEGFA, PDGFB, and TGFβ1 but displayed no expression of CD63, TSG101, and Alix. In vivo, the hASC-EV treatment prevented the formation of hypertrophic scars on postoperative day 28 and suppressed collagen deposition and myofibroblast aggregation. However, the EV-free medium did not prevent the formation of hypertrophic scars on the same time point and had little effect on collagen deposition and myofibroblast aggregation when compared with the control gr...

Background: Growing evidence has demonstrated that adipose-derived stem cell-derived extracellular vesicles enhance the survival of fat grafts and the browning of white adipose tissue. We evaluated whether supplementation with adipose-derived stem cell-derived extracellular vesicles promotes the survival and browning of fat grafts. Methods: Extracellular vesicles derived from adipose-derived stem cells were injected into fat grafts of C57BL/6 mice once per week until postgraft week 12. The grafts were collected and weighed after postgraft weeks 2, 4, and 12. The histological morphology, neovascularization, and the proportion of M2 macrophages of grafts were evaluated. The ability of extracellular vesicles to promote macrophage polarization and catecholamine secretion was detected. Whether the inducement of browning adipose differentiation is extracellular vesicles or the paracrine effect of M2 macrophages polarized by extracellular vesicles was also verified. Results: Grafts treated by extracellular vesicles derived from adipose-derived stem cells showed enhanced beige adipose regeneration with increased neovascularization, M2 macrophage proportion, and norepinephrine secretion at postgraft week 4. Increased retention and decreased fibrosis and necrosis were noted at postgraft week 12. The extracellular vesicles uptake by macrophages promoted M2 type polarization and catecholamine secretion while suppressing M1 type polarization. Of note, browning adipose differentiation with enhanced energy expenditure could be promoted only by the conditioned medium from extracellular vesicle–polarized M2 macrophages but not by extracellular vesicles themselves. Conclusions: Supplementation with extracellular vesicles derived from adipose-derived stem cells increases fat graft survival and browning by which extracellular vesicles–polarized M2 macrophages secrete catecholamines to promote beige adipose regeneration.

The number and size of resorption cavities in cancellous bone are believed to influence rates of bone loss, local tissue stress and strain and potentially whole bone strength. Traditional two-dimensional approaches to measuring resorption cavities in cancellous bone report the percent of the bone surface covered by cavities or osteoclasts, but cannot measure cavity number or size. Here we use three-dimensional imaging (voxel size 0.7 × 0.7 × 5.0 μm) to characterize resorption cavity location, number and size in human vertebral cancellous bone from nine elderly donors (7 male, 2 female, ages 47–80 years). Cavities were 30.10 ± 8.56 μm in maximum depth, 80.60 ± 22.23 *103 μm2 in surface area and 614.16 ± 311.93 *103 μm3 in volume (mean ± SD). The average number of cavities per unit tissue volume (N.Cv/TV) was 1.25 ± 0.77 mm−3. The ratio of maximum cavity depth to local trabecular thickness was 30.46 ± 7.03 % and maximum cavity depth was greater on thicker trabeculae (p < 0.05, r2 = 0.14). Half of the resorption cavities were located entirely on nodes (the intersection of two or more trabeculae) within the trabecular structure. Cavities that were not entirely on nodes were predominately on plate-like trabeculae oriented in the cranial-caudal (longitudinal) direction. Cavities on plate-like trabeculae were larger in maximum cavity depth, cavity surface area and cavity volume than cavities on rod-like trabeculae (p < 0.05). We conclude from these findings that cavity size and location are related to local trabecular microarchitecture.

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: Currently, there is a lack in therapy that promotes the reepithelialization of diabetic wounds as an alternative to skin grafting. Here, the authors hypothesized that extracellular vesicles from adipose-derived stem cells (ADSC-EVs) could accelerate wound closure through rescuing the function of keratinocytes in diabetic mice. Methods: The effect of ADSC-EVs on the biological function of human keratinocyte cells was assayed in vitro. In vivo, 81 male severe combined immune deficiency mice aged 8 weeks were divided randomly into the extracellular vesicle–treated diabetes group (n = 27), the phosphate-buffered saline–treated diabetes group (n = 27), and the phosphate-buffered saline–treated normal group (n = 27). A round, 8-mm-diameter, full-skin defect was performed on the back skin of each mouse. The wound closure kinetics, average healing time, reepithelialization rate, and neovascularization were evaluated by histological staining. Results: In vitro, ADSC-EVs improved proliferation, migration, and proangiogenic potential, and inhibited the apoptosis of human keratinocyte cells by suppressing Fasl expression with the optimal dose of 40 μg/mL. In vivo, postoperative dripping of ADSC-EVs at the dose of 40 μg/mL accelerated diabetic wound healing, with a 15.8% increase in closure rate and a 3.3-day decrease in average healing time. ADSC-EVs improved reepithelialization (18.2%) with enhanced epithelial proliferation and filaggrin expression, and suppressed epithelial apoptosis and Fasl expression. A 2.7-fold increase in the number of CD31-positive cells was also observed. Conclusion: ADSC-EVs improve diabetic wound closure and angiogenesis by enhancing keratinocyte-mediated reepithelialization and vascularization. Clinical Relevance Statement: ADSC-EVs could be developed as a regenerative medicine for diabetic wound care.

ecellularized adipose tissue represents a promising scaffold for adipose tissue engineering. Multiple decellular techniques have been demonstrated for decellularized adipose tissue preparation, for manufacture into scaffold materials with different properties, such as powder, sponge, or hydrogel. [1][2][3] Thermosensitive hydrogel liquefied at 4°C and solidified at 37°C was considered the most suitable material for softtissue filling because of its ability to be injected subcutaneously, its minimal invasiveness, and its plasticity for the irregular shape of soft-tissue defects. 4 However, inadequate angiogenesis and adipogenesis limits the recellularization and fat formation after decellularized adipose tissue grafting. 3,5,6 Adipose tissue regulates angiogenesis and adipogenesis through paracrine factors. 7,8 Previous studies have shown that adipose liquid extract, isolated by mechanical protocol from lipoaspirate,

Atopic dermatitis is defined as an intensely systemic inflammation among skin diseases. Exosomes derived from adipose-derived stem cells may be a novel cell-free therapeutic strategy for atopic dermatitis treatment. This study aims to elucidate the possible underlying mechanism of adipose-derived stem cells-exosomes harboring microRNA-147a in atopic dermatitis pathogenesis. BALB/c mice treated with Dermatophagoides farinae extract/2,4-dinitrochlorobenzene were defined as a mouse model of atopic dermatitis, either with inflamed HaCaT cells and HUVECs exposed with TNF-α/IFN-γ stimulation were applied for a cell model of atopic dermatitis. The concentrations of IL-1β and TNF-α in the supernatants were examined by ELISA. Cell viability and migration were assessed by MTT and Transwell assay. The apoptosis was examined using flow cytometry and TUNEL staining. The tube formation assay was employed to analyzed angiogenesis. The molecular regulations among miR-147a, MEF2A, TSLP and VEGFA were confirmed using luciferase reporter assay, either with ChIP. microRNA-147a was markedly downregulated in the serum and skin samples of atopic dermatitis mice, of which overexpression remarkably promoted HaCaT cell proliferation, meanwhile inhibiting inflammatory response and cell apoptosis. microRNA-147a in adipose-derived stem cells was subsequently overexpressed, and exosomes (Exos-miR-147a mimics) were collected. Functionally, exos-microRNA-147a mimics attenuated TNF-α/IFN-γ-induced HaCaT cell inflammatory response and apoptosis, and suppressed HUVECs angiogenesis. Encouraging, molecular interaction experiments revealed that exosomal microRNA-147a suppressed TNF-α/IFN-γ-induced HUVECs angiogenesis by targeting VEGFA, and exosomal microRNA-147a repressed HaCaT cells inflammatory injury through the MEF2A-TSLP axis. Mechanistically, exosomal microRNA-147a repressed pathological angiogenesis and inflammatory injury during atopic dermatitis progression by targeting VEGFA and MEF2A-TSLP axis. microRNA-147a-overexpressing adipose-derived stem cells-derived exosomes suppressed pathological angiogenesis and inflammatory injury in atopic dermatitis by targeting VEGFA and MEF2A-TSLP axis.