SCUBE3 Exerts a Tumor-Promoting Effect in Tongue Squamous Cell Carcinoma by Promoting CEBPA Binding to the CCL2 Promoter.

Tongue squamous cell carcinoma (TSCC) is the main pathologic subtype of oral cancer, and the current therapeutic effect is far from satisfactory. The signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) has been shown to be a tumor-promoting factor in several malignancies. However, little is known about the role of SCUBE3 in TSCC. In this study, we identified that SCUBE3 was highly expressed in TSCC. Clinically, high expression of SCUBE3 was positively associated with tumor stage and T stage of TSCC. Functionally, SCUBE3 silence remarkably restrained cell proliferation, migration, and invasion, induced apoptosis as well as cell cycle arrest in G2-phase, and weakened the tumorigenicity of TSCC cells in vivo. Mechanistically, SCUBE3 promoted the direct binding of CCAAT enhancer binding protein alpha (CEBPA) to C-C motif chemokine ligand 2 (CCL2) promoter in TSCC cells. Interestingly, CCL2 overexpression partially reversed the inhibitory effect of SCUBE3 deficiency on TSCC cell viability and migration. Moreover, STAT3 signaling contributed to CCL2-mediated phenotypes in TSCC cells. IMPLICATIONS: Our data revealed a tumor-promoting role for SCUBE3 in TSCC via the CEBPA/CCL2/STAT3 axis, which provided new insight into novel potential therapeutic target for TSCC.

An experimental study of the vascular embolism caused by recombinant type III collagen implants and hyaluronic acid.

BACKGROUND: There are significant differences in the reported incidence of vascular complications that result from the injection of different soft tissue fillers. This study aimed to compare the risk of vascular embolism after recombinant type III collagen implants and hyaluronic acid (HA) injection into arteries. METHODS: Different concentrations of recombinant type III collagen and Restylane were injected into the central ear artery of rabbits, to construct an immediate embolization model. We screened for vascular recanalization and tissue necrosis at 30 min, 1 day, and 7 days after injection, and histopathology examination was processed on Day 7. RESULTS: At 30 min after injection, complete recanalization of the central ear artery was observed in 17 rabbits in the C1 group while none in the HA group. On Day 1 after injection, complete recanalization of the CEA main trunk was observed in all rabbits in the collagen group while 50% in the HA group. There was a significant difference between the C1 group and the HA group in terms of vascular recanalization and skin necrosis. CONCLUSION: Under the present experimental conditions, the risk of causing vascular embolism was much lower with collagen than with Restylane. Different doses of collagen at different injection rates have the same safety profile.

Application of decellularization-recellularization technique in plastic and reconstructive surgery.

ABSTRACT: In the field of plastic and reconstructive surgery, the loss of organs or tissues caused by diseases or injuries has resulted in challenges, such as donor shortage and immunosuppression. In recent years, with the development of regenerative medicine, the decellularization-recellularization strategy seems to be a promising and attractive method to resolve these difficulties. The decellularized extracellular matrix contains no cells and genetic materials, while retaining the complex ultrastructure, and it can be used as a scaffold for cell seeding and subsequent transplantation, thereby promoting the regeneration of diseased or damaged tissues and organs. This review provided an overview of decellularization-recellularization technique, and mainly concentrated on the application of decellularization-recellularization technique in the field of plastic and reconstructive surgery, including the remodeling of skin, nose, ears, face, and limbs. Finally, we proposed the challenges in and the direction of future development of decellularization-recellularization technique in plastic surgery.

Targeting the Glycolytic Enzyme PGK1 to Inhibit the Warburg Effect: A New Strategy for Keloid Therapy.

BACKGROUND: Aerobic glycolysis (the Warburg effect) may play an important role in keloid pathogenesis, which may be aggravated by the hypoxic microenvironment in keloids. Phosphoglycerate kinase 1 (PGK1), a key glycolytic enzyme, is essential for cellular aerobic glycolysis, but its role in keloid formation remains unknown. This study aimed to detect PGK1 expression in keloid tissue and investigate the effects of inhibiting PGK1 expression on keloid fibroblasts (KFbs) under hypoxia and normoxia. METHODS: Normal skin and keloid samples were separated into two parts, one was used for immunohistochemistry, and one for primary cell culture. PGK1 tissue expression was detected by immunohistochemistry. Reverse-transcriptase polymerase chain reaction and Western blotting were used to detect PGK1, GLUT1, LDHA, and COL1 expression, and glucose uptake and lactate production were detected with a microplate reader. Cell proliferation and apoptosis were investigated with IncuCyte and flow cytometry. Cell migration and invasion were detected with Transwell assays. Glycolytic function was explored with the Seahorse XF96 system. RESULTS: Immunohistochemistry showed PGK1 overexpression in keloid tissue compared with normal skin tissue ( P < 0.05). Consistently, PGK1 expression was significantly higher in KFbs than in normal skin fibroblasts (NFbs), and hypoxia stimulated PGK1 expression in KFbs and NFbs ( P < 0.05). PGK1 knockdown significantly inhibited KFb glycolysis, proliferation, migration, invasion, glucose consumption, and lactate production ( P < 0.05). Furthermore, GLUT1, LDHA, and COL1 expression was decreased in KFbs compared with NFbs ( P < 0.05). In addition, suppressing PGK1 may mediate the PI3K/AKT pathway to down-regulate GLUT1, LDHA, and COL1 expression ( P < 0.05). CONCLUSIONS: These findings provide new evidence that suppressing PGK1, inhibiting glycolysis, reduces KFb proliferation, migration, invasion, and type I collagen expression. Targeting PGK1 to inhibit the Warburg effect may be a new therapeutic strategy for keloids. CLINICAL RELEVANCE STATEMENT: This article may provide new suggestions into the pathogenesis and treatment of keloids. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Extracellular vesicles derived from lipoaspirate fluid promote fat graft survival.

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.

Exosomes from adipose-derived stem cells and application to skin wound healing.

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.

Altered glucose metabolism and cell function in keloid fibroblasts under hypoxia.

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.

Differentiation potential and mRNA profiles of human dedifferentiated adipose cells and adipose­derived stem cells from young donors.

Dedifferentiated adipose cells (DAs) and adipose­derived 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 23­30 years) with a physiological BMI (19.1­23.9 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 6­7 passages, cell proliferation assays, phenotypic assessment, differentiation assays and high­throughput 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.

Associations of LRP5 Gene With Bone Mineral Density, Bone Turnover Markers, and Fractures in the Elderly With Osteoporosis.

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.

Sandstorm weather is a risk factor for mortality in ischemic heart disease patients in the Hexi Corridor, northwestern China.

Ischemic heart disease (IHD) is one of the leading causes of mortality worldwide. Moreover, the effects of air pollution have been associated with several cardiovascular diseases (CVDs). The relationship between sandstorm weather and IHD is unknown. The Hexi Corridor is located in northwestern China and is a typical desert region comprising a large area of desert with a high incidence of sandstorms. This study aimed to explore the association between sandstorm weather and IHD-related mortality in this area. We acquired meteorological data of sandstorm weather from 2006 to 2015 from the Gansu Meteorological Bureau, and data regarding deaths due to IHD in five cities within the Hexi Corridor were collected from the death registration system of the Center for Disease Control of Gansu during the same period. Two other cities with few sandstorm events were selected as control regions. The time series method of the generalized additive model (GAM) was used to assess the association between sandstorm weather and IHD-related mortality in the Hexi Corridor. The results showed that the frequency of sandstorms in the Hexi Corridor was higher than that in the control regions (5.48% vs 1.64%, P < 0.01), and IHD-related mortality was correspondingly higher than that in the control regions (56.42/100,000 vs 45.62/100,000, P < 0.01). After stratification by gender, age, and urban/rural residence, a significant difference in IHD-related mortality was also noted (P < 0.05). Significant associations were found between sandstorm weather and IHD-related mortality, and the relative risk (RR) increased with an increasing number of days of sandstorm weather. According to the monthly and annual analyses, the mortality rate corresponded to sandstorm frequency. Our data suggest a positive association between sandstorm weather and IHD-related mortality in the Hexi Corridor of Gansu Province. The underlying mechanism requires further study.

Effects of photoelectric therapy on proliferation and apoptosis of scar cells by regulating the expression of microRNA-206 and its related mechanisms.

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/cm2 laser, and the high-energy group was irradiated by 20 J/cm2 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 < .05). The higher the energy was, the greater the upregulation of apoptosis was. Apoptosis and cell migration increased (P < .05). The expressions of microRNA-206, MMP-9, and Bax were upregulated, while the expressions of mTOR, Bcl-2, and cyclin D1 were downregulated. To sum up, laser irradiation can significantly inhibit the proliferation of HSF cells, affect cell cycle, and increase cell apoptosis and migratory ability.

Stromal vascular fraction promotes migration of fibroblasts and angiogenesis through regulation of extracellular matrix in the skin wound healing process.

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.

Exosomes from Adipose-Derived Stem Cells (ADSCs) Overexpressing miR-21 Promote Vascularization of Endothelial Cells.

In the past few years, exosomes released from adipose-derived stem cells (abbreviated as ADSCs) have shown promises to provide therapeutic benefits in the fields of regenerative medicine. miRNAs, existing in exosomes, are endogenous, small noncoding RNAs that play important roles in a variety of cellular functions and tumor development. Emerging evidences have indicated that miR-21 is one of the important miRNAs associated with tumor angiogenesis. In this study, we identified the role of exosomes from ADSCs overexpressing miR-21 in regulating/promoting vascularization of endothelial cells. Experimental data indicated an elevated miR-21 level in exosomes released by ADSCs overexpressing miR-21. In vitro matrigel angiogenesis assay showed that exosomes secreted by ADSCs overexpressing miR-21 significantly promoted the vascularization of HUVEC cells (an endothelial cell line). Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) revealed an upregulation of HIF-1α, VEGF, SDF-1, p-Akt, p-ERK1/2 and downregulation of PTEN in response to miR-21 overexpression, indicating that miR-21 enriched exosomes induced angiogenesis through Akt and ERK activation and also HIF-1α and SDF-1 expression. Our work suggests that exosomes from ADSCs that overexpressing miR-21 can potentially promote vascularization and therefore the transplantation of exosomes from their culture may be suitable for clinical effort in regenerative medicine.

Parathyroid hormone (PTH) promotes ADSC osteogenesis by regulating SIK2 and Wnt4.

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.

Risk Comparison of Filler Embolism Between Polymethyl Methacrylate (PMMA) and Hyaluronic Acid (HA).

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 .

Effect of 810 nm Near-Infrared Laser on Revascularization of Ischemic Flaps in Rats.

OBJECTIVE: To investigate the effect of 810 nm near-infrared (NIR) laser on the revascularization of ischemic flaps. BACKGROUND: 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. MATERIALS AND METHODS: 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/cm2 daily. The control flaps were covered by stainless steel to avoid laser irradiation. We observed the viability of the flaps. The flaps underwent Hematoxylin and Eosin (H&E) staining for the observation of histomorphology, immunohistochemical staining of factor VIII for the capillary count, α-smooth muscle actin for the small arterial count, and vascular endothelial growth factor for the integrated optical density (OD) of the positive stained color. RESULTS: The irradiated flaps showed significantly better flap survival than the control flaps. H&E 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. CONCLUSIONS: PBMT with 810 nm NIR laser could enhance ischemic flap revascularization and increase flap viability.

Basic and Clinical Evidence of an Alternative Method to Produce Vivo Nanofat.

BACKGROUND: Fat grafting technologies are popularly used in plastic and reconstructive surgery. Due to its size limitation, it is hard to directly inject untreated fat tissue into the dermal layer. Nanofat, which was introduced by Tonnard, solves this problem by mechanically emulsifying fat tissue. However, the viability of the cells was greatly destroyed. In this study, we reported a new method by "gently" digesting the fat tissue to produce viable adipocytes, progenitors, and stromal stem cells using collagenase I digestion and centrifugation. This was named "Vivo nanofat". METHODS: Human liposuction aspirates were obtained from five healthy female donors with mean age of 28.7 ± 5.6 years. Colony-forming assay, flow cytometry analysis, and adipogenic and osteogenic induction of the adherent cells from the Vivo nanofat were used to characterize the adipose mesenchymal stem cells (MSCs). To investigate in vivo survival, we respectively injected Vivo nanofat and nanofat subcutaneously to the back of 8-week-old male BALB/c nude mice. Samples were harvested 2 days, 2 weeks, and 4 weeks postinjection for measurement, hematoxylin and eosin staining, and immunostaining. RESULTS: Our results showed that the Vivo nanofat contained a large number of colony-forming cells. These cells expressed MSC markers and had multi-differentiative potential. In vivo transplantation showed that the Vivo nanofat had lower resorption ratio than that of nanofat. The size of the transplanted nanofat was obviously smaller than that of Vivo nanofat 4 weeks postinjection (0.50 ± 0.17 cm vs. 0.81 ± 0.07 cm, t = -5783, P = 0.01). CONCLUSION: Vivo nanofat may serve as a cell fraction injectable through a fine needle; this could be used for cosmetic applications.

In vitro Flow Perfusion Maintaining Long-term Viability of the Rat Groin Fat Flap: A Novel Model for Research on Large-scale Engineered Tissues.

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.

Metabolic reprogramming in keloid fibroblasts: Aerobic glycolysis and a novel therapeutic strategy.

Keloids, tumor-like fibroproliferative cutaneous lesions, were reported in metabolic disturbance. However, the metabolic character remains unclear. The purpose of this study is to determine if glycolytic reprogramming is important for the pathogenesis of keloids and to assess the inhibition potential of glycolysis in keloid treatment. An intracellular metabolic profile assay was used to compare metabolic phenotypes between normal skin fibroblasts and keloid fibroblasts (NFs and KFs). Our data indicated that KFs underwent reprogramming of their metabolic phonotype from oxidative phosphorylation to aerobic glycolysis (Warburg effect) with augmented glycolysis and glycolytic capacity. Both gene and protein assays showed that the expression of glycolytic enzymes was upregulated in KFs compared to NFs. Our data showed higher glucose influx and lactate production in KFs compared to NFs. Furthermore, the proliferation of KFs was suppressed in a dose-dependent and time-dependent manner after inhibition of glycolysis with 2-deoxy-glucose (2-DG). Taken together, these findings suggested that keloids underwent a reprogrammed metabolic phenotype of aerobic glycolysis. This was essential for keloid hyperplasia, and glycolytic inhibitors might provide a potential treatment for keloids.

Upregulated periostin promotes angiogenesis in keloids through activation of the ERK 1/2 and focal adhesion kinase pathways, as well as the upregulated expression of VEGF and angiopoietin­1.

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<0.01) and a weak correlation was observed using CD105 staining (r=0.251; P<0.01). Conditioned medium from keloid fibroblasts (KFs) promoted the migration and tube formation of human umbilical vein endothelial cells (HUVECs) compared with normal fibroblasts and this effect may have been abrogated by the short hairpin RNA knockdown of periostin. Treatment with recombinant human periostin promoted the migration and tube formation of HUVECs by activating the extracellular signal­regulated kinase 1/2 and focal adhesion kinase signaling pathway. In addition, periostin increased the secretion of vascular endothelial growth factor and angiopoietin­1 in the KFs. In conclusion, these data suggested that upregulation in the level of periostin may promote angiogenesis directly and indirectly in keloids and may be a key factor in keloid development. Periostin may, therefore, be a promising therapeutic target in the treatment of keloids and other angioproliferative diseases.