LncRNA RPARP-AS1 promotes the progression of osteosarcoma cells through regulating lipid metabolism.

Osteosarcoma (OS) is a highly malignant tumor, and its dysregulated lipid metabolism is associated with tumorigenesis and unfavorable prognosis. Interestingly, long noncoding RNAs (lncRNAs) have emerged as pivotal regulators of lipid metabolism, exerting notable impacts on tumor proliferation. Nevertheless, the involvement of RPARP-AS1, a novel lipid metabolism-associated lncRNA, remains unexplored in the context of OS. This study aims to identify functionally relevant lncRNAs impacting OS proliferation and lipid metabolism and seeks to shed light on the upstream regulatory mechanisms governing lipogenic enzyme activity. Based on comprehensive bioinformatic analysis and the establishment of a risk model, we identified seven lncRNAs significantly associated with clinical characteristics and lipid metabolism-related genes in patients with OS. Among these, RPARP-AS1 was selected for in-depth investigation regarding its roles in OS proliferation and lipid metabolism. Experimental techniques including RT-qPCR, Western blot, cell viability assay, assessment, and quantification of free fatty acids (FFAs) and triglycerides (TGs) were utilized to elucidate the functional significance of RPARP-AS1 in OS cells and validate its effects on lipid metabolism. Manipulation of RPARP-AS1 expression via ectopic expression or siRNA-mediated knockdown led to alterations in epithelial-mesenchymal transition (EMT) and expression of apoptosis-associated proteins, thereby influencing OS cell proliferation and apoptosis. Mechanistically, RPARP-AS1 was found to augment the expression of key lipogenic enzymes (FABP4, MAGL, and SCD1) and potentially modulate the Akt/mTOR pathway, thereby contributing to lipid metabolism (involving alterations in FFA and TG levels) in OS cells. Collectively, our findings establish RPARP-AS1 as a novel oncogene in OS cells and suggest its role in fostering tumor growth through the enhancement of lipid metabolism.

Activation of moderate autophagy promotes survival of fat graft.

Clinically unpredictable retention following fat grafting remains outstanding problems because of the unrevealed mechanism of grafted fat survival. The role of autophagy, a process to maintain cellular homeostasis through recycling cellular debris, has yet been to be reported in fat grafting. This study aims to improve the survival of fat grafting through the autophagy. First, the relationship between cell death and autophagy in the early stage of fat grafting was evaluated through immunostaining, RNA sequencing, and western blot. Next, rapamycin, an autophagic agonist, was used for the culturing of adipose-derived stem cells and adipocytes during ischemia. Cell death, autophagy, and reactive oxygen species (ROS) were assayed. Finally, rapamycin was used to assist fat grafting in nude mice. The results demonstrated that the peak of cell death at the early stage of fat grafting was accompanied by a decrease in autophagy. In vitro, during ischemia, 25 nM was confirmed as the optimal dose of rapamycin that reduces cell death with enhanced autophagy and mitophagy, improved mitochondrial quality as well as decreased ROS accumulation. In vivo, promoted mitophagy, alleviated oxidative stress, and decreased cell apoptosis of rapamycin-treated fat grafts were observed in the early stage. In addition, rapamycin increased the survival of fat grafts with increased neovascularization and reduced fibrosis. We suggested that moderate autophagy induced by rapamycin contribute to enhanced ischemic tolerance and long term survival of fat grafts through mitochondrial quality control.

Association of Monocyte to Lymphocyte, Neutrophil to Lymphocyte, and Platelet to Lymphocyte Ratios With Non-Healing Lower Extremity Ulcers in Patients With Type 2 Diabetes.

OBJECTIVE: Diabetic ulcers are a prevalent complication of diabetes mellitus and represent one of the most complex and severe complications that can occur in diabetic patients. Most existing studies have separately examined the neutrophil-to-lymphocyte ratio (NLR) prognostic value or the platelet-to-lymphocyte ratio (PLR). However, to our knowledge, no study has evaluated the relationship between the monocyte-to-lymphocyte ratio (MLR) and non-healing lower extremity ulcers (NHLU) in patients. This study explored the association between 3 hematological parameters (MLR, NLR, and PLR) and the risk of non-healing ulceration in patients with type 2 diabetes (T2D). METHODS: A cross-sectional study was performed using data from the National Health and Nutrition Examination Survey (NHANES) spanning 1999 to 2004. The primary outcome variable was NHLU status, determined by patients' self-reported responses to the question, "Have you had an ulcer or sore on your leg or foot that took more than four weeks to heal?" Logistic regression examined the relationships between MLR, NLR, PLR, and NHLU. Stratified analyses were also conducted based on age, gender, hemoglobin (HGB) level, and body mass index (BMI). RESULTS: In the multivariate regression models, after adjusting for age, sex, race/ethnicity, marital status, poverty income ratio (PIR), BMI, HGB, family history of diabetes, and low-density lipoprotein (model 3), the odds ratios (ORs) of MLR and NLR were 1.21 (1.09-1.33) and 1.02 (1.01-1.03), respectively. However, the association was no longer statistically significant for the NLR (OR = 1.0002, 95% CI: 0.99-1.0005, P = .137). In the subgroup analysis, the effect sizes of MLR and NLR on the presence of NHLU were stable in all subgroups (all P > .05). CONCLUSIONS: After adjusting for confounding variables, MLR and NLR were significantly increased in T2D participants with NHLU. They may play a significant role in monitoring T2D patients during follow-up visits.

Exosomes with overexpressed miR 147a suppress angiogenesis and infammatory injury in an experimental model of atopic dermatitis.

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-1ß 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.

A Novel Preoperative Design to Address Dermatochalasis in Asian Upper Blepharoplasty.

BACKGROUND: The preoperative design for the amount of skin excision of the upper eyelid is a common procedure in Asian blepharoplasty, but there lack of an effective method addressing dermatochalasis to acquire esthetically pleasing results. Our aim was to propose an alternative technique to determine the skin excision combined with the esthetics of eyes for correcting skin laxity and therefore to create attractive double eyelids. MATERIALS AND METHODS: The preoperative invaginating-simulating design combined with esthetic criteria for determining the amount of excised skin were performed during blepharoplasty. The Strasser grading system and the Global Aesthetic Improvement Scale were evaluated by analyzing the preoperative and 6-month-postoperative photographs. RESULTS: One hundred forty-two patients were included. The general outline of the "optimal incision" took on a knife in shape with a mean of 2.2±3.32 points of Strasser grading score. A total of 130 of 142 patients (91.5%) were judged as "good results" and 12 of 142 patients (8.5%) were judged as "mediocre". The Global Aesthetic Improvement Scale showed a significant cosmetic improvement with the result of 89.4%(127 of 142 patients) for "very much improved", 7.8% for "much improved" and other patients for "improved". No severe complications were observed. CONCLUSIONS: This preoperative design approach has been proven to be effective to address dermatochalasis (especially for lateral hooding) by simulating the expected appearance of a double eyelid combined with esthetic criteria of eyes simultaneously, which can contribute to achieving upper-lid rejuvenation and beautiful and natural outcomes.

Adipose-Derived Stem Cell Extracellular Vesicles Improve Wound Closure and Angiogenesis in Diabetic Mice.

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.

Extracellular Vesicles Isolated From Hypoxia-Preconditioned Adipose-Derived Stem Cells Promote Hypoxia-Inducible Factor 1α-Mediated Neovascularization of Random Skin Flap in Rats.

BACKGROUND: Random flaps are widely used for wound repair. However, flap necrosis is a serious complication leading to the failure of operation. Our previous study demonstrated a great proangiogenic potential of hypoxia-treated adipose-derived stem cells-extracellular vesicles (HT-ASC-EVs). Thus, we aim to evaluate the effect of HT-ASC-EVs in the survival and angiogenesis of random skin flap in rats. METHODS: Adipose-derived stem cells-extracellular vesicles were respectively isolated from adipose-derived stem cell culture medium of 3 donors via ultracentrifugation. The expression of hypoxia-inducible factor 1α (HIF-1α) and proangiogenic potential of HT-ASC-EVs and ASC-EVs were compared by co-culturing with human umbilical vein endothelial cells. Forty male Sprague-Dawley rats were randomly divided into 3 group (n = 10/group). A 9 × 3-cm random skin flap was separated from the underlying fascia with both sacral arteries sectioned on each rat. The survival and angiogenesis of flaps treated by ASC-EVs or HT-ASC-EVs were also compared. Laser Doppler flowmetry and immunohistochemistry were used to evaluate skin perfusion and angiogenesis of skin flaps on postoperative day 7. RESULTS: Hypoxia-treated adipose-derived stem cells-extracellular vesicles further improve the proliferation, migration, tube formation with upregulated HIF-1α, and VEGF expression of human umbilical vein endothelial cells in vitro, compared with ASC-EVs. In vivo, postoperatively injecting HT-ASC-EVs suppressed necrosis rate (29.1 ± 2.8% vs 59.2 ± 2.1%) and promoted the angiogenesis of skin flap including improved skin perfusion (803.2 ± 24.3 vs 556.3 ± 26.7 perfusion unit), increased number of CD31-positive cells, and upregulated expression of HIF-1α in vascular endothelium on postoperative day 7, compared with ASC-EVs. CONCLUSIONS: Intradermal injecting HT-ASC-EVs improve the survival of random skin flap by promoting HIF-1α-mediated angiogenesis in rat model.

Extracellular Vesicles Derived from Adipose-Derived Stem Cells Accelerate Diabetic Wound Healing by Suppressing the Expression of Matrix Metalloproteinase-9.

BACKGROUND: The healing of diabetic wounds is poor due to a collagen deposition disorder. Matrix metalloproteinase-9 (MMP-9) is closely related to collagen deposition in the process of tissue repair. Many studies have demonstrated that extracellular vesicles derived from adipose-derived stem cells (ADSC-EVs) promote diabetic wound healing by enhancing collagen deposition. OBJECTIVE: In this study, we explored whether ADSC-EVs could downregulate the expression of MMP-9 in diabetic wounds and promote wound healing by improving collagen deposition. The potential effects of ADSC-EVs on MMP-9 and diabetic wound healing were tested both in vitro and in vivo. METHODS: We first evaluated the effect of ADSC-EVs on the proliferation and MMP-9 secretion of HaCaT cells treated with advanced glycation end product-bovine serum albumin (AGE-BSA) using CCK-8, western blot and MMP-9 enzyme-linked immunosorbent assay(ELISA). Next, the effects of ADSC-EVs on healing, re-epithelialisation, collagen deposition, and MMP-9 concentration in diabetic wound fluids were evaluated in an immunodeficient mouse model via MMP-9 ELISA and haematoxylin and eosin, Masson's trichrome, and immunofluorescence staining for MMP-9. RESULTS: In vitro, ADSC-EVs promoted the proliferation and MMP-9 secretion of HaCaT cells. In vivo, ADSC-EVs accelerated diabetic wound healing by improving re-epithelialisation and collagen deposition and by inhibiting the expression of MMP-9. CONCLUSION: ADSC-EVs possess the potential of healing of diabetic wounds in a mouse model by inhibiting downregulating MMP-9 and improving collagen deposition. Thus, ADSC-EVs are a promising candidate for the treatment of diabetic wounds.

Preparing Adipogenic Hydrogel with Neo-Mechanical Isolated Adipose-Derived Extracellular Vesicles for Adipose Tissue Engineering.

BACKGROUND: Subcutaneous transplantation of decellularized adipose tissue was capable of recellularization during soft tissue repair. However, further improvements are required to promote angiogenesis and adipogenesis. Here, the authors proposed a neo-mechanical protocol to isolate adipose tissue-derived extracellular vesicles (ATEVs) through lipoaspirate as a mediator for both angiogenesis and adipogenesis, and prepared ATEV-rich decellularized adipose tissue hydrogel for adipose tissue engineering. METHODS: Adipose liquid extract and lipid-devoid adipose tissue were extracted through homogenization and repeated freeze and thaw cycles. ATEVs were isolated from adipose liquid extract by ultracentrifugation. Decellularized adipose tissue hydrogel was prepared by optimized decellularization of lipid-devoid adipose tissue. The optimum dose of ATEVs for angiogenesis and adipogenesis was estimated by co-culturing with vascular endothelial cells and 3T3-L1 cells, then mixed with the hydrogel. ATEV-enriched hydrogel was injected subcutaneously into the back of severe combined immunodeficiency mice, and then subjected to supplementary injection of ATEVs on postoperative day 14. ATEV-free decellularized adipose tissue hydrogel was injected as control. The newly formed tissue samples were harvested at postoperative weeks 2, 4, and 8 and subjected to volume measurement, hematoxylin and eosin staining, and immunofluorescence (CD31 and perilipin) staining. RESULTS: The optimum dose of ATEVs for promoting angiogenesis and adipogenesis was 50 µg/ml. The newly formed tissue mediated by ATEV-enriched hydrogel had increased volume well as improved angiogenesis and adipogenesis at postoperative week 4 and 8. CONCLUSION: ATEV-enriched adipogenic hydrogel promotes enhanced angiogenesis and adipogenesis and could serve as a promising biomaterial for adipose tissue engineering.

Extracellular Vesicles Derived From Human Adipose-Derived Stem Cell Prevent the Formation of Hypertrophic Scar in a Rabbit Model.

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 ß 1 (TGFß1). 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 TGFß1. The EV-free medium had a high expression of VEGFA, PDGFB, and TGFß1 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 group. CONCLUSIONS: Our study suggests that hASCs are associated with preventive scar formation therapy because of paracrine EVs rather than cytokines. A local injection of hASC-EVs during wound healing efficiently prevented hypertrophic scar formation, which may have a clinically beneficial antiscarring effect.

Supplementation with Extracellular Vesicles Derived from Adipose-Derived Stem Cells Increases Fat Graft Survival and Browning in Mice: A Cell-Free Approach to Construct Beige Fat from White Fat Grafting.

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.

Extracellular vesicles derived from human adipose-derived stem cells promote the exogenous angiogenesis of fat grafts via the let-7/AGO1/VEGF signalling pathway.

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 co-cultured 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 pre-treatment. 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.

[Effect of circulating estrogen level on the outcome of free fat grafting in nude mice].

OBJECTIVE: To investigate the effect of circulating estrogen level on the outcome of free fat grafting in nude mice. METHODS: Eighteen female nude mice aged 6-8 weeks (weighing, 20-25 g) were randomly divided into 3 groups ( n=6). The nude mice in the ovariectomized group were treated with ovariectomy. The nude mice in the high estrogen group and the normal estrogen group only made the same incision to enter the peritoneum without ovariectomy. The nude mice in the high estrogen group were given the estradiol (0.2 mg/g) every 3 days for 30 days. The other two groups were given the same amount of PBS every 3 days. At 30 days after operation, the tail vein blood of nude mice in 3 groups were detected by estradiol ELISA kit, and the free fat (0.3 mL) donated by the females was injected into the sub-scalp of nude mice. After 8 weeks of fat grafting, the samples were taken for gross observation and weighing, and the prepared slices were stained with HE staining, CD31-perilipin fluorescence staining, immunohistochemical staining of uncoupling protein 1 (UCP1), and immunofluorescence staining of estrogen receptor α. The diameter of adipocytes and vascular density of adipose tissue were measured. The mRNA expressions of UCP1 and estrogen receptor α were detected by realtime fluorescence quantitative PCR (qRT-PCR). RESULTS: All nude mice survived during experiment. ELISA test showed that the concentration of estradiol significantly decreased in the ovariectomized group and increased in the high estrogen group compared with the normal estrogen group ( P<0.05). At 8 weeks after fat grafting, the graft volume from large to small was ovariectomized group, normal estrogen group, and high estrogen group. There was significant difference in wet weight between the ovariectomized group and high estrogen group ( P<0.05). Section staining showed that compared with the normal estrogen group, the adipocytes in the ovariectomized group were larger, the expression of peri-lipoprotein was weaker, the vascular density decreased, and the expressions of UCP1 was negative, and the estrogen receptor α positive cells reduced. The above observation results in the high estrogen group were contrary to those in the ovariectomized group. There were significant differences in the diameter of adipocytes, the vascular density of adipose tissue, the number of the estrogen receptor α positive cells between groups ( P<0.05). The results of qRT-PCR showed that the mRNA expressions of UCP1 and estrogen receptor α significantly increased in the high estrogen group and decreased in the ovariectomized group compared with the normal estrogen group, and the differences were significant ( P<0.05). CONCLUSION: The level of circulating estrogen has a significant effect on the outcome of free fat grafting in nude mice. Low estrogen level leads to hypertrophy of graft adipocytes, while high estrogen level leads to the production of a large amount of beige fat and high vascular density in fat grafts, which may be related to the activation of estrogen receptor α on adipocytes.

[Construction of tissue engineered adipose by human adipose tissue derived extracellular vesicle combined with decellularized adipose tissues scaffold].

OBJECTIVE: To explore the possibility of constructing tissue engineered adipose by adipose tissue derived extracellular vesicles (hAT-EV) combined with decellularized adipose tissue (DAT) scaffolds, and to provide a new therapy for soft tissue defects. METHODS: The adipose tissue voluntarily donated by the liposuction patient was divided into two parts, one of them was decellularized and observed by HE and Masson staining and scanning electron microscope (SEM). Immunohistochemical staining and Western blot detection for collagen type Ⅰ and Ⅳ and laminin were also employed. Another one was incubated with exosome-removed complete medium for 48 hours, then centrifuged to collect the medium and to obtain hAT-EV via ultracentrifugation. The morphology of hAT-EV was observed by transmission electron microscopy; the nanoparticle tracking analyzer (NanoSight) was used to analyze the size distribution; Western blot was used to analyse membrane surface protein of hAT-EV. Adipose derived stem cells (ADSCs) were co-cultured with PKH26 fluorescently labeled hAT-EV, confocal fluorescence microscopy was used to observe the uptake of hAT-EV by ADSCs. Oil red O staining was used to evaluate adipogenic differentiation after hAT-EV and ADSCs co-cultured for 15 days. The DAT was scissored and then injected into the bilateral backs of 8 C57 mice (6-week-old). In experimental group, 0.2 mL hAT-EV was injected weekly, and 0.2 mL PBS was injected weekly in control group. After 12 weeks, the mice were sacrificed, and the new fat organisms on both sides were weighed. The amount of new fat was evaluated by HE and peri-lipoprotein immunofluorescence staining to evaluate the ability of hAT-EV to induce adipogenesis in vivo. RESULTS: After acellularization of adipose tissue, HE and Masson staining showed that DAT was mainly composed of loosely arranged collagen with no nucleus; SEM showed that no cells and cell fragments were found in DAT, and thick fibrous collagen bundles could be seen; immunohistochemical staining and Western blot detection showed that collagen type Ⅰ and Ⅳ and laminin were retained in DAT. It was found that hAT-EV exhibited a spherical shape of double-layer envelope, with high expressions of CD63, apoptosis-inducible factor 6 interacting protein antibody, tumor susceptibility gene 101, and the particle size of 97.9% hAT-EV ranged from 32.67 nmto 220.20 nm with a peak at 91.28 nm. Confocal fluorescence microscopy and oil red O staining showed that hAT-EV was absorbed by ADSCs and induced adipogenic differentiation. In vivo experiments showed that the wet weight of fat new organisms in the experimental group was significantly higher than that in the control group ( t=2.278, P=0.048). HE staining showed that the structure of lipid droplets in the experimental group was more than that in the control group, and the collagen content in the control group was higher than that in the experimental group. The proportion of new fat in the experimental group was significantly higher than that in the control group ( t=4.648, P=0.017). CONCLUSION: DAT carrying hAT-EV can be used as a new method to induce adipose tissue regeneration and has a potential application prospect in the repair of soft tissue defects.

[Effects of adipose-derived stem cell released exosomes on wound healing in diabetic mice].

OBJECTIVE: To investigate the effects of adipose-derived stem cell released exosomes (ADSC-Exos) on wound healing in diabetic mice. METHODS: The ADSCs were isolated from the adipose tissue donated by the patients and cultured by enzymatic digestion. The supernatant of the 3rd generation ADSCs was used to extract Exos (ADSC-Exos). The morphology of ADSC-Exos was observed by transmission electron microscopy. The membrane-labeled proteins (Alix and CD63) were detected by Western blot, and the particle size distribution was detected by nanoparticle tracking analyzer. The fibroblasts were isolated from the skin tissue donated by the patients and cultured by enzymatic digestion. The 5th generation fibroblasts were cultured with PKH26-labeled ADSC-Exos, and observed by confocal fluorescence microscopy. The effects of ADSC-Exos on proliferation and migration of fibroblasts were observed with cell counting kit 8 (CCK-8) and scratch method. Twenty-four 8-week-old Balb/c male mice were used to prepare a diabetic model. A full-thickness skin defect of 8 mm in diameter was prepared on the back. And 0.2 mL of ADSC-Exos and PBS were injected into the dermis of the experimental group ( n=12) and the control group ( n=12), respectively. On the 1st, 4th, 7th, 11th, 16th, and 21st days, the wound healing was observed and the wound healing rate was calculated. On the 7th, 14th, and 21st days, the histology (HE and Masson) and CD31 immunohistochemical staining were performed to observe the wound structure, collagen fibers, and neovascularization. RESULTS: ADSC-Exos were the membranous vesicles with clear edges and uniform size; the particle size was 40-200 nm with an average of 102.1 nm; the membrane-labeled proteins (Alix and CD63) were positive. The composite culture observation showed that ADSC-Exos could enter the fibroblasts and promote the proliferation and migration of fibroblasts. Animal experiments showed that the wound healing of the experimental group was significantly faster than that of the control group, and the wound healing rate was significantly different at each time point ( P<0.05). Compared with the control group, the wound healing of the experimental group was better. There were more microvessels in the early healing stage, and more deposited collagen fibers in the late healing stage. There were significant differences in the length of wound on the 7th, 14th, and 21st days, the number of microvessels on the 7th and 14th days, and the rate of deposited collagen fibers on the 14th and 21st days between the two groups ( P<0.05). CONCLUSION: ADSC-Exos can promote the wound healing in diabetic mice by promoting angiogenesis and proliferation and migration of fibroblasts and collagen synthesis.

[Effect of adipose-derived stem cell derived exosomes on angiogenesis after skin flap transplantation in rats].

OBJECTIVE: To investigate the effect of adipose-derived stem cell derived exosomes (ADSC-Exos) on angiogenesis after skin flap transplantation in rats. METHODS: ADSCs were isolated and cultured by enzymatic digestion from voluntary donated adipose tissue of patients undergoing liposuction. The 3rd generation cells were observed under microscopy and identified by flow cytometry and oil red O staining at 14 days after induction of adipogenesis. After cells were identified as ADSCs, ADSC-Exos was extracted by density gradient centrifugation. And the morphology was observed by transmission electron microscopy, the surface marker proteins (CD63, TSG101) were detected by Western blot, and particle size distribution was measured by nanoparticle size tracking analyzer. Twenty male Sprague Dawley rats, weighing 250-300 g, were randomly divided into ADSC-Exos group and PBS group with 10 rats in each group. ADSC-Exos (ADSC-Exos group) and PBS (PBS group) were injected into the proximal, middle, and distal regions of the dorsal free flaps with an area of 9 cm×3 cm along the long axis in the two groups. The survival rate of the flap was measured on the 7th day, and then the flap tissue was harvested. The tissue morphology was observed by HE staining, and mean blood vessel density (MVD) was measured by CD31 immunohistochemical staining. RESULTS: ADSCs were identified by microscopy, flow cytometry, and adipogenic induction culture. ADSC-Exos was a round or elliptical membrane vesicle with clear edge and uniform size. It has high expression of CD63 and TSG101, and its size distribution was 30-200 nm, which was in accordance with the size range of Exos. The distal necrosis of the flaps in the ADSC-Exos group was milder than that in the PBS group. On the 7th day, the survival rate of the flaps in the ADSC-Exos group was 64.2%±11.5%, which was significantly higher than that in the PBS group (31.0%±6.6%; t=7.945, P=0.000); the skin appendages in the middle region of the flap in the ADSC-Exos group were more complete, the edema in the proximal region was lighter and the vasodilation was more extensive. MVD of the ADSC-Exos group was (103.3±27.0) /field, which was significantly higher than that of the PBS group [(45.3±16.2)/field; t=3.190, P=0.011]. CONCLUSION: ADSC-Exos can improve the blood supply of skin flaps by promoting the formation of neovascularization after skin flap transplantation, thereby improve the survival rate of skin flaps in rats.

[Progress of mesenchymal stem cells derived exosomes in wound repair].

OBJECTIVE: To summarize the research progress of mesenchymal stem cells derived exosomes (MSCs-EXOs) in wound repair in recent years. METHODS: The literature about the role of MSCs-EXOs in wound repair at home and abroad was extensively consulted. The mechanism of MSCs-EXOs in wound repair and its clinical application prospects were summarized and analyzed. RESULTS: MSCs-EXOs can inhibit early inflammatory reaction, promote angiogenesis, proliferation, and migration of epithelial cells, regulate collagen synthesis, and inhibit scar proliferation in the later stage of wound healing. Compared with MSCs, MSCs-EXOs have many advantages, such as high stability, easy storage, non-tumorigenicity, no proliferation, easy quantitative use, and so on. It has broad clinical application prospects. CONCLUSION: MSCs-EXOs can promote wound repair and hopefully develop into a clinical product to promote the repair of acute or chronic wounds.