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.

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.

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.

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.

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.

[The effects of conditioned medium from keloid fibroblasts under hypoxia on angiogenesis].

OBJECTIVE: To observe the effects of conditioned medium from keloid fibroblasts under hypoxia on angiogenesis, and to investigate the role of hypoxic microenvironment in invasive growth of keloid. METHODS: Primary keloid fibroblasts and human umbilical endothelial cells (HUVEC) were cultured as conventional method. Keloid fibroblasts were cultured either in a hypoxic incubator (2% O2) for 48 h or in a normoxic incubator (20% O2) as control. Then those cell culture mediums were collected and mixed with endothelial cell medium by the proportion of 1:1 as conditioned medium. The mRNA and secreted protein of pro-angiogenic factors such as vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and periostin of keloid fibroblasts under hypoxia were detected by real time PCR and ELISA. The proliferation, migration and invasion, tube formation of HUVEC cultured with conditioned medium were evaluated by CCK-8 assay, Transwell assay and matrigel tube formation assay, respectively. RESULTS: Hypoxia increased the expression of VEGF, Ang-1 and periostin in both mRNA (increased by 75%, 43% and 118% respectively, P < 0.05) and secreted protein (increased by 30.2%, 14.2% and 19.5% respectively, P < 0.05) levels; the proliferations of HUVEC in hypoxic conditioned medium in 1, 2 and 3 d were 0.67 +/- 0.07, 0.84 +/- 0.09 and 1.08 +/- 0.10 respectively, which were higher compared to those in control group (0.52 +/- 0.08, 0.72 +/- 0.10 and 0.91 + 0.14, P < 0.05); the numbers of migration, invasion and tube formation of HUVEC were (73.2 +/- 8.9), (56.3 +/- 12.5), (9.66 +/- 1.96) cells/HP, which were higher compared to those in control group [(59.0 +/- 8.0), 35.5 +/- 8.5), (6.5 +/- 1.87) cells/HP, P < 0.05]. CONCLUSIONS: Hypoxia increases the expression of pro-angiogenic factors of keloid fibroblasts, and its conditioned medium under hypoxia could promote angiogenesis. The results suggest hypoxic microenvironment may play a significant role in the invasive growth of keloid by inducing angiogenesis.

Increased periostin expression affects the proliferation, collagen synthesis, migration and invasion of keloid fibroblasts under hypoxic conditions.

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 αvß3 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.

[Variations in expressions of periostin and related factors in early stage of wound healing and scar remodeling in rats].

OBJECTIVE: To investigate the expressions of periostin (PN), angiopoietin-1 (Ang-1), vascular epithelial growth factor (VEGF) and fetal liver kinase-1 (Flk-1) during the processes of scar formation and modulation in rat cutaneous wounds and probe into their roles in wound healing and scaring. METHODS: Eighty-two male Sprague-Dawley (SD) rats were randomly divided into 10 groups with 8-9 rats in each group. Two 2 cm×2 cm full-thickness excisional wounds in the back were created in each rat. The wound surface was observed, and the healing area was measured. The pathological change was observed after hematoxylin and eosin (HE) staining. The expressions of PN, Ang-1, VEGF and Flk-1 in wound surface scar at 4-8 weeks were determined with immunohistochemistry. The expressions of PN, Ang-1 and VEGF were determined by Western blotting. The normal skin was served as control. RESULTS: HE staining showed that the wound surface tissue had healed with epithelization at 4-8 weeks. Immunohistochemistry results showed that there was no significant difference in Flk-1 expression between wound surface tissue and normal skin. The PN expression (A value/µm(2)) in wound surface tissue was significantly lower than that in normal skin at 5 weeks (2.43±0.44 vs. 4.24±0.50, P<0.05), and the expression of Ang-1 and VEGF (A value/µm(2)) at 4, 5, 6, 8 weeks was significantly lower than that in normal skin (Ang-1: 3.51±0.93, 3.10±0.57, 2.77±0.59, 2.77±1.26 vs. 4.89±0.48; VEGF: 1.76±0.68, 1.75±0.49, 1.99±0.42, 1.94±0.86 vs. 4.86±1.63, all P<0.05). In wound surface scar, PN and Flk-1 positive signal was found in cell, and the Ang-1 and VEGF positive signal in extracellular matrix. Western blotting data demonstrated that the expressions of PN, Ang-1 and VEGF peaked at the 10th day after excision with increases to 7.90-22.56 folds compared with normal skin (PN: 2.45±1.51 vs. 0.31±0.19, Ang-1: 18.43±15.20 vs. 1.53±1.42, VEGF: 6.09±4.66 vs. 0.27±0.13, P<0.05 or P<0.01), and then followed with a decrease. CONCLUSIONS: PN, Ang-1, VEGF and Flk-1 are transiently overexpressed in early stage of full-thickness cutaneous wound healing in rats. Their expressions vary in wounds and scars. They participate in the healing of full-thickness cutaneous wounds together and may be essential for the proliferation stage during wound healing.

Biological characteristics of human adipose-derived stem cells and their response to periostin in vitro.

BACKGROUND: Many studies on periostin have focused on its role in tumors and vascular reconstruction. However, the effect of periostin on stem cell function remains unclear. The aim of this study was to enhance vitality in adipose-derived stem cells (ADSCs), the effect of periostin on the function of ADSCs was observed. METHODS: Human ADSCs (hADSCs) were isolated from human adipose tissue by collagenase I digestion and collected in multi-periods for in vitro culture. CD29, CD34, CD44, CD45 and CD105 were detected by flow cytometry. In addition, directed differentiation of hADSCs was induced using adipogenic, osteogenic and chondrogenic induction mediums. The induced morphological changes were observed using oil red O, Alizarin red and alcian blue staining. Periostin was administered to hADSCs in an acidic environment. The treatments of cells were divided into three groups: a periostin group (P); an acidic control group (A); a normal group (N). Then the resulting cell proliferation and migration were detected using a Cell Counting Kit-8 (CCK-8) and a transwell chamber assay, respectively. RESULTS: The detection rates of CD29, CD44, CD105, CD34 and CD45 were 98.89%, 93.73%, 86.99%, 0.19% and 0.16%. The specific staining of cells was positive after induction culture. The mean absorbance of the cells in group P and A at 12 hours were 16.67% and 22.22% greater than group N, respectively (P < 0.01). The mean absorbance of cells from group P was 20.00% greater than that of group A at 48 hours (P < 0.05). The mean number of migratory cells per visual field in group A was 50.38% lower than that in group N (P < 0.05). The migratory cell number in group P was 119.98% greater than that in group A (P < 0.05). CONCLUSIONS: The acidic environment impacted hADSC proliferation and inhibited cell migration. However, periostin was able to promote the proliferation and migration of hADSCs despite the acidic environment.

A snapshot of gene expression signatures generated using microarray datasets associated with excessive scarring.

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.

[The expressions of periostin and the related factors during healing process of full-thickness cutaneous wound in rat].

OBJECTIVE: To observe the expression of osteoblast-specific factor 2 (periostin, PN), angiopoietin-1 (Ang-1), vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 [VEGFR-2/fetal liver kinase-1 (FLK-1)] in wound surface and its peripheral skin, and their effects on wound healing in rats. METHODS: Forty-eight Sprague-Dawley (SD) rats were randomly divided into six groups, with 8 rats in each group. An area of 2 cm×2 cm full-thickness skin was excised on both sides of the back of rats. Specimens from wounds were obtained on 1, 4, 7, 10, 14, 21 days after operation, and histological evaluation and immunohistochemical staining of PN, Ang-1, VEGF and FLK-1 were made to determine their expression levels. Normal skin specimens were obtained as normal controls. RESULTS: The expressions of PN, Ang-1, VEGF and FLK-1 were significantly increased in wound surface after operation. Compared with the skin of normal controls, the expression of PN in the tissues of wound increased by 234.4% on the 1st day, and then increased continuously up to 597.9% on the 7th day (reaching the peak) after operation, followed by a decrease, the increase rate was 280.9% on the 21st day, and still remained at a high level (all P < 0.05). The expression of Ang-1 in the tissue of wound increased by 128.1% on the 1st day and 327.5% on the 4th day (reaching the peak), and then, it was gradually decreased. The increase rate was only 80.5% on the 14th day and it rose slightly later (all P < 0.05). The expression of VEGF in the tissues of wound reached the peak (165.8%) on the 7th day. Then it decreased with a slight fluctuation (all P < 0.05). The expression of FLK-1 in the tissues of wound was increased by 56.1% on the 1st day, and the level remained. It reached the peak by an increase of 70.1% on the 7th day (both P < 0.05). Then, it was lowered after the 10th day (all P > 0.05). CONCLUSIONS: The expressions of PN, Ang-1, VEGF, FLK-1 were obviously increased during healing of skin wound, with different peaking time and expressing rates. The increase in expression of PN showed the longest duration and highest peak value. The PN, Ang-1, VEGF, FLK-1 all play a role in the wound healing process, and PN might play an important role during the healing process of a full-thickness cutaneous wound.

[Application of acellular matrix in cartilage tissue engineering].

OBJECTIVE: To review the application of and the research progress on acellular matrix (ACM) in cartilage tissue engineering. METHODS: Related literatures both at home and abroad were retrospected and analyzed. RESULTS: Many researchers improved the properties of cartilage ACM scaffold through co-application of solution diosmosis method, freeze-drying method and physical and chemical cross-linking method etc., and the experimental results of applying cartilage ACM scaffold for the construction of tissue engineered cartilage were closely related to the properties of ACM. CONCLUSION: ACM has a wide application prospect for the construction of tissue engineered cartilage, and further in-depth studies are needed to improve its property.