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.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.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.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.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.
To investigate the effect of adipose-derived stem cell derived exosomes (ADSC-Exos) on angiogenesis after skin flap transplantation in rats.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.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].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.探讨脂肪干细胞来源外泌体（adipose-derived stem cell derived exosomes，ADSC-Exos）对大鼠皮瓣移植后血管新生的影响。.取抽脂术患者自愿捐赠脂肪组织，采用酶消化法分离培养 ADSCs；取第 3 代细胞光镜观察形态，流式细胞术鉴定细胞表面标志物，成脂诱导培养 14 d 后油红 O 染色观察。细胞鉴定为 ADSCs 后，采用密度梯度离心法提取 ADSC-Exos；透射电镜观察形态，Western blot 检测膜表面标志蛋白（CD63、TSG101），纳米颗粒跟踪分析仪测量其粒径分布。取 20 只雄性 SD 大鼠，体质量 250～300 g，随机分为 ADSC-Exos 组和 PBS 组，每组 10 只。两组大鼠背部沿长轴方向制作面积为 9 cm×3 cm 随意皮瓣后，分别于近端、中间和远端区域注射 ADSC-Exos（ADSC-Exos 组）或 PBS（PBS 组）。术后大体观察皮瓣成活情况，第 7 天测算皮瓣成活率后切取皮瓣组织，HE 染色观察组织形态，CD31 免疫组织化学染色测定皮瓣微血管密度（mean blood vessel density，MVD）。.光镜、流式细胞术及成脂诱导培养鉴定培养细胞为 ADSCs。ADSC-Exos 为边缘清晰、大小形态均匀的圆形或椭圆形膜性囊泡，高表达标志蛋白 CD63 和 TSG101，粒径分布在 30～200 nm，符合外泌体粒径范围。术后两组皮瓣远端均出现不同程度坏死表现，但 ADSC-Exos 组程度较 PBS 组轻；第 7 天 ADSC-Exos 组皮瓣成活率为 64.2%±11.5%，显著大于 PBS 组的 31.0%±6.6%（t=7.945，P=0.000）；中间区域皮肤附属器官更完整，近端区域组织水肿程度轻、血管扩张更多。ADSC-Exos 组 MVD 为（103.3±27.0）个/视野，显著多于 PBS 组（45.3±16.2）个/视野（t=3.190，P=0.011）。.ADSC-Exos 可通过促进皮瓣移植后新生血管的形成，改善皮瓣血供，进而提高大鼠皮瓣成活率。.
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.
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.
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 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.
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.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.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.ADSC-EVs improve diabetic wound closure and angiogenesis by enhancing keratinocyte-mediated reepithelialization and vascularization.ADSC-EVs could be developed as a regenerative medicine for diabetic wound care.
To investigate the effects of adipose-derived stem cell released exosomes (ADSC-Exos) on wound healing in diabetic mice.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.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).ADSC-Exos can promote the wound healing in diabetic mice by promoting angiogenesis and proliferation and migration of fibroblasts and collagen synthesis.探讨脂肪干细胞来源外泌体（adipose-derived stem cell released exosomes，ADSC-Exos）对糖尿病小鼠创面愈合的影响。.取患者自愿捐赠脂肪组织，采用酶消化法分离培养 ADSCs，并于第 3 代细胞上清液提取 Exos（ADSC-Exos）；透射电镜观察 ADSC-Exos 形态，Western blot 检测膜表面标志性蛋白 Alix、CD63，纳米颗粒跟踪分析仪检测粒径分布。取患者自愿捐赠皮肤组织，采用酶消化法分离培养成纤维细胞。将 PKH26 标记的 ADSC-Exos 与第 5 代成纤维细胞培养，共聚焦荧光显微镜观察其能否进入细胞，采用细胞计数试剂盒 8（cell counting kit 8， CCK-8）及划痕法观察 ADSC-Exos 对成纤维细胞增殖及迁移的影响。取 24 只 8 周龄 Balb/c 雄性小鼠制备糖尿病模型后，背部制备直径 8 mm 全层皮肤缺损创面，实验组（ n=12）及对照组（ n=12）创面真皮层分别注射 0.2 mL ADSC-Exos 及 PBS。第 1、4、7、11、16、21 天大体观察创面愈合情况，计算创面愈合率；第 7、14、21 天取材，行组织学（HE 及 Masson）及 CD31 免疫组织化学染色，观察创面结构、胶原纤维及新生血管情况。.ADSC-Exos 为边缘清晰、大小分布均匀的膜性囊泡；粒径为 40～200 nm，平均 102.1 nm；膜表面标志性蛋白 Alix、CD63 均呈阳性。ADSC-Exos 与成纤维细胞复合培养观察示，ADSC-Exos 可进入成纤维细胞胞内，并能促进成纤维细胞增殖及迁移。动物实验显示，实验组小鼠背部创面愈合明显快于对照组，各时间点创面愈合率差异均有统计学意义（ P<0.05）。与对照组比较，实验组创面结构愈合更好，愈合前期新生微血管更多，愈合后期胶原纤维沉积更多；其中，第 7、14、21 天创面缺损长度，第 7、14 天微血管数量，第 14、21 天胶原纤维沉积百分比，组间差异均有统计学意义（ P<0.05）。.ADSC-Exos 通过促进创面血管新生以及成纤维细胞增殖、迁移和胶原合成来促进糖尿病小鼠创面愈合。.
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.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.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.ATEV-enriched adipogenic hydrogel promotes enhanced angiogenesis and adipogenesis and could serve as a promising biomaterial for adipose tissue engineering.
Abstract 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.
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.
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