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Extracellular vesicles (EVs) are specific subcellular vesicles released by cells under various environmental conditions. Tumescent liposuction is a commonly used procedure in plastic surgery practice. In the present study, we aimed to extract EVs derived from lipoaspirate fluid (LF-EVs) and characterize them using transmission electron microscopy, nanoparticle tracking analysis, and western blotting. The global profiles of proteins and microRNAs from LF-EVs were identified, strongly suggesting a potential regulatory function of LF-EVs. In addition, we investigated the effects and mechanisms of LF-EVs on fat graft survival. Cell functional tests showed that LF-EVs promoted the proliferation, migration, and tube structure formation of human umbilical vein endothelial cells. LF-EVs also promoted the adipogenic differentiation of adipose tissue-derived stem cells. The results of animal experiments showed that the average weights of fat grafts in the LF-EVs-treated group were significantly higher than those in the control group. Histologically, there was less fibrosis, fewer cysts, and increased fat tissue survival in the LF-EVs group. Further investigations of angiogenic and adipogenic factors revealed that LF-EVs also promoted angiogenesis and exerted a pro-adipogenic effect in vivo. Our findings will help to elucidate the functions of LF-EVs and provide a reference dataset for future translational studies.

Background A refractory wound is a typical complication of diabetes and is a common outcome after surgery. Current approaches have difficulty in improving wound healing. Recently, non-expanded stromal vascular fraction (SVF), which is derived from mature fat, has opened up new directions for the treatment of refractory wound healing. The aim of the current study is to systematically investigate the impact of SVF on wound healing, including the rate and characteristics of wound healing, ability of fibroblasts to migrate, and blood transport reconstruction, with a special emphasis on their precise molecular mechanisms. Methods SVF was isolated by digestion, followed by filtration and centrifugation, and then validated by immunocytochemistry, a MTS proliferation assay and multilineage potential analysis. A wound model was generated by creating 6-mm-diameter wounds, which include a full skin defect, on the backs of streptozocin-induced hyperglycemic mice. SVF or human adipose-derived stem cell (hADSC) suspensions were subcutaneously injected, and the wounds were characterized over a 9-day period by photography and measurements. A scratch test was used to determine whether changes in the migratory ability of fibroblasts occurred after co-culture with hADSCs. Angiogenesis was observed with human umbilical vein endothelial cells. mRNA from fibroblasts, endotheliocyte, and skin tissue were sequenced by high-throughput RNAseq, and differentially expressed genes, and pathways, potentially regulated by SVF or hADSCs were bioinformatically analyzed. Results Our data show that hADSCs have multiple characteristics of MSC. SVF and hADSCs significantly improved wound healing in hyperglycemic mice. hADSCs improve the migratory ability of fibroblasts and capillary structure formation in HUVECs. SVF promotes wound healing by focusing on angiogenesis and matrix remodeling. Conclusions Both SVF and hADSCs improve the function of fibroblast and endothelial cells, regulate gene expression, and promote skin healing. Various mechanisms likely are involved, including migration of fibroblasts, tubulogenesis of endothelial cells through regulation of cell adhesion, and cytokine pathways.

Background The incidence of vascular complications varies among different fillers. The main purpose of this study was to compare the risk of embolism between PMMA (Artecoll) and hyaluronic acid (HA, Restylane) after artery injection. Methods Rabbit ears were injected via the central artery with 0.1 ml PMMA (group A), 0.1 ml HA (group B), 0.2 ml PMMA (group C), or 0.2 mL HA (group D), respectively. The formation of transparent emboli was monitored right after injection. Tissue necrosis and histopathological changes were analyzed on day 7. Results With 0.1 ml injected volume, PMMA was dispersed within a few minutes and only 5% of the injected ears had mild necrosis on day 7, while HA tended to form obvious transparent emboli, an indication of blood vessel clotting, and 60% of injected ears showed necrosis on day 7. With 0.2 ml injected volume, PMMA had a risk of complete blood vessel clotting in between 0.1 ml PMMA group and 0.1 ml HA group, and 30% of injected ears had necrosis; in contrast, 100% of 0.2 ml HA-injected ears showed transparent emboli and necrosis. The necrosis areas were significantly increased in the HA groups compared with PMMA groups at the same injection volumes. HA injection also caused dilation of small blood vessels. Conclusion At the same injection volume, PMMA had less risk of embolism compared with HA. With increased injection volume, there were increased risks of embolism and necrosis for both PMMA and HA. No Level Assigned 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. Electronic supplementary material The online version of this article (10.1007/s00266-019-01320-w) contains supplementary material, which is available to authorized users.

Skin wound healing is an intractable problem that represents an urgent clinical need. To solve this problem, a large number of studies have focused on the use of exosomes (EXOs) derived from adipose‐derived stem cells (ADSCs). This review describes the mechanisms whereby ADSCs‐EXOs regulate wound healing and their clinical application. In the wound, ADSCs‐EXOs modulate immune responses and inflammation. They also promote angiogenesis, accelerate proliferation and re‐epithelization of skin cells, and regulate collagen remodelling which inhibits scar hyperplasia. Compared with ADSCs therapeutics, ADSCs‐EXOs have highly stability and are easily stored. Additionally, they are not rejected by the immune system and have a homing effect and their dosage can be easily controlled. ADSCs‐EXOs can improve fat grafting and promote wound healing in patients with diabetes mellitus. They can also act as a carrier and combined scaffold for treatment, leading to scarless cutaneous repair. Overall, ADSCs‐EXOs have the potential to be used in the clinic to promote wound healing., This review describes the mechanisms whereby exosomes (EXOs) derived from adipose‐derived stem cells (ADSCs) regulate wound healing and their clinical application. In the wound, ADSCs‐EXOs modulate immune responses and inflammation, promote angiogenesis, accelerate proliferation and re‐epithelization of skin cells and regulate collagen remodelling, which inhibits scar hyperplasia. ADSCs‐EXOs have the potential to be used in the clinic, as they can improve fat grafting, promote wound healing of diabetic patients and act as a carrier and combined scaffold for treatment, leading to scarless cutaneous repair.

Dedifferentiated adipose cells (DAs) and adipose‑derived stem cells (ADSCs) are two of the primary types of stem cells derived from adipose tissue, which have been reported to possess similar characteristics, but also exhibit unique phenotypic and functional advantages. However, several reports have described inconsistent results regarding their differences in multilineage differentiation function. Moreover, to the best of our knowledge, there are no studies assessing their myogenic ability, or the differences in the transcriptome between the two cell types derived from lipoaspirates via tumescent liposuction from the same donors. The aim of the present study was to compare the properties and expression profiles of these cell types. Subcutaneous adipose tissue of three female patients (aged 23‑30 years) with a physiological BMI (19.1‑23.9 kg/m2) were obtained during tumescent liposuction of the abdomen or the thigh. The stromal vascular fraction and mature adipocytes were obtained via collagenase digestion, and ADSCs and DAs were cultured successively. To determine the differences between DAs and ADSCs after 6‑7 passages, cell proliferation assays, phenotypic assessment, differentiation assays and high‑throughput RNA sequencing (seq) were used. Similar cell morphologies, proliferation dynamics, surface markers and transcriptome expression profiles were observed between the DAs and ADSCs. Whilst there were notable individual differences in the osteogenic, lipogenic, chondrogenic and myogenic abilities of the DAs and ADSCs, it was difficult to determine their differentiation potential based only on the cell source. Interestingly, the myogenic ability was relatively stronger in cells with relatively weaker lipogenic ability. Only 186 differentially expressed genes between the two groups were identified using RNAseq. Several of these genes were involved in biological functions such as transcription regulation, protein translation regulation, cytokine interactions and energy metabolism regulation. The results of the present study suggested a similar functional potential of DAs and ADSCs from young donors undergoing tumescent liposuction operation in regeneration areas and the balance of the differentiative ability of the same cell populations. These data may provide a foundation for further clinical administration of stem cells derived from adipose tissues in therapy.

Objective: As an aberrant response to cutaneous wound healing, keloid scarring is a challenge to cure, and its clinical management remains unsatisfactory. The purpose of this study was to investigate whether mitochondrial function and morphology were impaired in keloid pathology. Approach: Keloid fibroblasts (KFs) and normal skin fibroblasts (NFs) were isolated and cultured from excised samples. A real-time extracellular flux analyzer was used to monitor the oxygen consumption rate for evaluating the mitochondrial respiratory function. Real-time PCR or Western blot was performed to detect the levels of mRNAs or proteins, respectively, including those associated with the tricarboxylic acid cycle, mitochondrial fission and fusion, and mitochondrial biogenesis. The reactive oxygen species (ROS) level and the mitochondrial mass were measured by flow cytometry. Mitochondrial ultrastructure was observed by transmission electron microscopy. Results: Compared with NFs, KFs showed reduced basal oxidative respiration (p

Objective: The study aimed to explore the associations of rs4988300 and rs634008 in the low-density lipoprotein receptor–related protein 5 (LRP5) gene with bone mineral density (BMD), bone turnover markers (BTM), and fractures in elderly patients with osteoporosis (OP).Methods: Our study included 328 unrelated OP patients with or without fractures. Genomic DNA was extracted for genotyping. BTM levels were assessed by electrochemiluminescence (ECL). Dual-energy X-ray absorptiometry (DXA) was employed to measure BMD in the lumbar spine (LS) and proximal femur. Basic features between the OP and fracture groups were analyzed using the t-test. The Chi-square test was performed to analyze the differences in allele and genotype frequencies. The associations of single-nucleotide polymorphisms (SNPs) with BMD and BTM in the subgroups were investigated by the analysis of covariance (ANCOVA) adjusted for confounding factors.Results: In both females and males, individuals with fractures exhibited higher BTM levels and lower BMD values than those with OP (P < 0.05). The allele and genotype frequencies of rs4988300 in the subgroups were significantly different (P < 0.05). In both females and males suffering from OP, participants with rs4988300 GG or rs634008 TT presented lower procollagen I N-terminal propeptide (PINP) levels (P < 0.05). Women with OP carrying rs4988300 GG exhibited lower BMD values at FN and TH (P < 0.05). In both females and males with fractures, individuals carrying rs4988300 GG genotype or rs634008 TT genotype exhibited lower PINP levels and BMD values at FN and TH than those with other genotypes (P < 0.05).Conclusions: Rs4988300 and rs634008 polymorphisms in the LRP5 gene are associated with bone phenotypes in the elderly with OP or fractures.

To investigate the effect of 810 nm near-infrared (NIR) laser on the revascularization of ischemic flaps.It has long been proved that photobiomodulation therapy (PBMT) improves the blood supply of flaps. NIR laser improves the treatment of hypodermis-located lesions and of flap survival, but basic research on the use of 810 nm NIR laser for ischemic flap revascularization is still lacking.We prepared two symmetrical long random-pattern flaps on the backs of 60 rats. Each flap was 6 cm long, 1 cm wide, and 1 cm to the middle line. The flaps were divided into an irradiated flap group and an internal control group. The irradiated flaps underwent postoperative 810 nm laser therapy with the energy density of 11.30 J/cmThe irradiated flaps showed significantly better flap survival than the control flaps. HE staining showed that the irradiated flaps had clear tissue structure and little inflammatory cell infiltration. The control flaps demonstrated comparatively worse results. Vascular endothelial growth factor staining showed that the difference in integrated OD between the irradiated flaps and the control flaps was not statistically significant. α-smooth muscle actin and factor VIII staining showed significantly greater numbers of arterioles and capillaries in the irradiated flaps than the control flaps after 4 days of irradiation.PBMT with 810 nm NIR laser could enhance ischemic flap revascularization and increase flap viability.

Human skin fibroblast (HSF) cells were irradiated with different energy lasers to detect cell proliferation, apoptosis, and expression of microRNA‐206 and protein, and to further summarise the therapeutic effect of laser on scar cells. Human scar cell line HSF cells were cultured in three groups. The control group was not irradiated by laser, the low‐energy group was irradiated by 10 J/cm(2) laser, and the high‐energy group was irradiated by 20 J/cm(2) laser. After irradiation, HSF cells were cultured for 20 hours. Cell proliferation was detected by MTT assay. Cell cycle and apoptosis were detected by flow cytometry. Transwell migration assay was used to detect cell migratory ability. Reverse transcription polymerase chain reaction (RT‐PCR) was used to detect miR‐206 and mTOR gene levels. The levels of MMP‐9, Bax, Bcl‐2, cyclin D1, and mTOR signalling pathway proteins were detected by Western blotting assays. The results showed that after laser irradiation, the proliferation of cells decreased, and the difference between the control group and the experimental group was significant (P

Developing methods for regenerating bones and uncovering the molecular mechanism underlying bone formation have great significance to human health. In the last decade, people have been using adipose-derived stem cells (ADSCs), that are capable of multilineage differentiation, to reconstruct defected bones. Uncovering the molecular mechanisms of the osteoblast differentiation of ADSCs will provide more understanding of ADSCs in the application of bone regeneration and perhaps new methods for osteoporosis treatment. Here we studied how parathyroid hormone (PTH1-34) acts on osteoinduced ADSCs to orchestrate bone formation and how Wnt4 signaling is involved in PTH-promoted bone formation from ADSCs. We found that PTH1-34 can phosphorylate SIK2, upregulate RANKL and downregulate SOST, thereby upregulating Wnt4 to promote the osteogenesis process of ADSCs. Though the knockdown of Wnt4 with shRNA interference barely affects the expression of upstream proteins (i.e., RANKL, SOST), it affects the expression of other downstream osteogenic proteins (i.e., Runx2, Osterix, and Osteocalcin), and then inhibit the osteogenesis process of ADSCs. Overall, PTH can affect the osteogenesis process of ADSCs by regulating SIK2 and Wnt4. We anticipate that this work will provide researchers with new insights into the bone regeneration with ADSCs.

Keloids exhibit metabolic reprogramming including enhanced glycolysis and attenuated oxidative phosphorylation. Hypoxia induces a series of protective responses in mammalian cells. However, the metabolic phenotype of keloid fibroblasts under hypoxic conditions remains to be elucidated. The present study aimed to investigate glycolytic activity, mitochondrial function and morphology, and the HIF1α and PI3K/AKT signaling pathways in keloid fibroblasts (KFB) under hypoxic conditions. Our results showed that hypoxia promoted proliferation, migration invasion and collagen synthesis and inhibited apoptosis in KFB. The mRNA levels, protein expressions and enzyme activities of glycolytic enzymes in KFB were higher than those in normal skin fibroblasts (NFB) under normoxia. Moreover, hypoxia remarkedly upregulated glycolysis in KFB. Decreased activities of mitochondrial complexes and abnormal mitochondria were detected in KFB under normoxic conditions and the damage was aggravated by hypoxia. An intracellular metabolic profile assay suggested hypoxia increased glycolytic parameters except glycolytic reserve but inhibited the key parameters of mitochondrial function apart from H+ leak. Protein levels of HIF1α and phosphorylation levels of the PI3K/AKT signaling pathway were upregulated in the context of 3% oxygen. Enhanced total reactive oxygen species (ROS), mitochondrial ROS (mitoROS) and antioxidant activities of KFB were observed in response to hypoxia. Additionally, autophagy was induced by hypoxia. Our data collectively demonstrated potentiated glycolysis and attenuated mitochondrial function under hypoxia, indicating that altered glucose metabolism regulated by hypoxia could be a therapeutic target for keloids.

Background: Large-scale muscle tissue engineering remains a major challenge. An axial vascular pedicle and perfusion bioreactor are necessary for the development and maintenance of large-scale engineered muscle to ensure circulation within the construct. We aimed to develop a novel experimental model of a large-scale engineered muscle flap from an existing rat groin fat flap. Methods: A fat flap based on the superficial inferior epigastric vascular pedicle was excised from rats and placed into a perfusion bioreactor. The flaps were kept in the bioreactor for up to 7 weeks, and transdifferentiation of adipose to muscle tissue could have taken place. This system enabled myogenic-differentiation medium flow through the bioreactor at constant pH and oxygen concentration. Assessment of viability was performed by an immunofluorescence assay, histological staining, a calcein-based live/dead test, and through determination of RNA quantity and quality after 1, 3, 5, and 7 weeks. Results: Immunofluorescence staining showed that smooth muscle around vessels was still intact without signs of necrosis or atrophy. The visual assessment of viability by the calcein-based live/dead test revealed viability of the rat adipose tissue preserved in the bioreactor system with permanent perfusion. RNA samples from different experimental conditions were quantified by spectrophotometry, and intact bands of 18S and 28S rRNA were detected by gel electrophoresis, indicating that degradation of RNA was minimal. Conclusions: Flow perfusion maintains the long-term viability of a rat groin engineered muscle flap in vitro, and a large-scale vascularized muscle could be engineered in a perfusion bioreactor. Key words: Culture Ex vivo; Flow Perfusion; Perfusion Bioreactor; Tissue Engineering

Periostin, a secreted extracellular matrix protein, is highly expressed in wound healing and in various types of human cancer and is involved in angiogenesis. Keloids, considered dermal benign tumors, are granulomatous lesions characterized by capillary proliferation. However, the underlying regulatory mechanism of angiogenesis in keloids remains to be elucidated. The present study aimed to examine the effect of periostin on angiogenesis in keloids. The expression of periostin was upregulated and the vessel density was higher in human keloids compared with normal tissue, observed following staining with CD31 and CD105. Periostin demonstrated a markedly positive correlation with blood vessel density, which was assessed using CD31 staining (r=0.711; P

Periostin, a secreted extracellular matrix protein, is involved in the wound healing and pathological process of various human cancers. Keloid scars are fibroproliferative tumor-like lesions and develop under local hypoxia. Using suppression subtractive hybridization, in a previous study, we found that periostin is overexpressed in keloids compared with hypertrophic scars. However, little is known about the regulation and function of periostin in keloids. In this study, we examined the effects of periostin on the bioactivity of keloid fibroblasts (KFs) in order to determine whether periostin is involved in hypoxia-stimulated keloid pathogenesis by measuring the expression levels of periostin in KFs cultured under hypoxic conditions. We also investigated the association between periostin and hypoxia-inducible factor-1α (HIF-1α). The mRNA, intracellular protein and secreted protein level of periostin was examined by RT-PCR (and quantitative PCR), western blot analysis and enzyme-linked immunosorbent assay (ELISA), respectively. We also used shRNA targeting periostin to knockdown its expression in the KFs. We report that hypoxia (2% O(2)) upregulates both HIF-1α and periostin expression in KFs. In addition, hypoxia-upregulated periostin expression was regulated by HIF-1α. The inhibition of periostin by short hairpin RNA decreased the hypoxia-stimulated proliferation, collagen synthesis, migration and invasion of KFs and altered the cell cycle, but did not affect apoptosis; treatment with recombinant human periostin protein reversed these effects. Periostin also activated the αvβ3 integrin-PI3K/Akt pathway in the KFs. These findings suggest that hypoxia initiates hyperplasia of KFs and increases periostin expression under hypoxic conditions; periostin is involved in the pathogenesis of keloids, which indicates that periostin may be a novel therapeutic target for keloids and other fibroproliferative disorders.

Objective The aim of this study was to examine the gene expression signatures of 2 types of excessive scarring, keloid and hypertrophic scar (HS), through the integrated bioinformatics analysis of multiple published gene expression profile datasets. Methods A literature search of microarray data published in focusing on keloid and HS was performed. The microarray data were reanalyzed to identify the common significantly dysregulated (CSD) genes. The experimental and statistical parameters of the studies were systematically evaluated to reveal any influences on the consensus findings among the studies. Overrepresentation analysis of gene ontology (GO) categories and pathways was used to explore the significantly dysregulated genes functionally associated with the pathogenesis of excessive scarring. Results Seven published microarray studies on keloid and 4 studies on HS were identified. A total of 64 CSD genes were identified in keloid; 9 upregulated CSD genes were identified in HS. The 48 consistently dysregulated genes that overlapped in least 1 keloid study and 1 HS study were defined as the CSD genes in excessive scarring. The differences in the variables associated with the study protocols and data management were systematically documented and evaluated. Activated GO categories and pathways related to skeletal development, binding, extracellular matrix-receptor interaction, and adhesion were found to have significance in excessive scarring, implying a common pathological basis for keloid and HS formation. Notably, the GO categories related to cancer and the TGF-beta signaling pathway were significantly enriched in keloids. Conclusions As gene signatures and molecular markers of excessive scarring, the identified CSD genes may be particularly relevant to disease pathogenesis and serve as new therapeutic targets.