Manipulating electrostatic field to control the distribution of bioactive proteins or polymeric microparticles on planar surfaces for guiding cell migration.

We report a general strategy to generate linear and circular gradients of active proteins or polymeric microparticles on planar surfaces by controlling the distribution of electrostatic field during electrohydrodynamic jet printing or electrospray process. Taking fibronectin as an example, we generated a circular gradient of fibronectin and investigated its effect on accelerating the migration of fibroblasts to suit for use in wound closure. In another demonstration, we created linear gradients of laminin in unidirectional and bidirectional patterns, respectively. We showed that such gradations significantly promoted the migration of human neuroblastoma cells with the increase of laminin content. When we changed fibronectin/laminin to electrosprayed poly(lactic-co-glycolic acid) (PLGA) microparticles, we found similar results in terms of guiding cell migration, except that the guidance cues varied from biological signal to topographic structure. Taken together, this method for generating linear/circular gradients of fibronectin/laminin and PLGA microparticles can be readily extended to different types of bioactive proteins and polymeric microparticles to suit wound closure, nerve repair, and related applications involving cell migration.

Harnessing biocompatible nanofibers and silver nanoparticles for wound healing: Sandwich wound dressing versus commercial silver sulfadiazine dressing.

Owing to the structural replication of native extracellular matrix, nonwoven mats of electrospun nanofibers have great potential for use in wound healing. Herein, we report the design and fabrication of a sandwich wound dressing to balance its antimicrobial activity and biocompatibility. This success mainly relies on the incorporation of silver nanoparticles (AgNPs) into electrospun nanofibers, together with the rational design of a sandwich structure for the dressing. The bottom layer was composed of hydrophilic nanofibers made from a blend of polycaprolactone (PCL) and gelatin (Gel). The top layer consisted of hydrophobic PCL nanofibers. AgNP-loaded PCL/Gel nanofibers were sandwiched between the two layers. When compared with a commercial silver sulfadiazine dressing, the designed wound dressing showed competitive antimicrobial properties, lower cell toxicity, and accelerated wound closure for mouse skin injury. By balancing the biocompatibility of electrospun nanofibers and the broad-spectrum antibacterial activity of AgNPs within a sandwich structure, the novel multifunctional wound dressing could be valuable for effective wound healing and related applications.

HOXC10 promotes growth and migration of melanoma by regulating Slug to activate the YAP/TAZ signaling pathway.

Homeobox C10 (HOXC10) has been reported to participate in various cancers. However, the involvement of HOXC10 in melanoma is still unknown. Here, we attempted to determine whether HOXC10 can affect the development of melanoma. We separated melanoma tissues and the matched tumor-adjacent normal tissues from melanoma patients, and examined HOXC10 expression in the melanoma cells and tissues. Comparing with the tumor-adjacent normal tissues, HOXC10 was up-regulated in melanoma tissues. Melanoma cells also displayed an up-regulation of HOXC10. Moreover, HOXC10 inhibition suppressed cell proliferation, clone formation and promoted apoptosis of melanoma cells. Knockdown of HOXC10 also retarded migration, invasion and epithelial-mesenchymal transition (EMT) in melanoma cells. Additionally, HOXC10 accelerated Slug expression by interacting with Slug, and activating the promoter of Slug. Slug activated the YAP/TAZ signaling pathway, which was reversed by HOXC10 silencing. The in vitro assays demonstrated that inhibition of HOXC10 significantly repressed tumor growth and lung metastasis of melanoma in mice by inhibiting Slug and YAP/TAZ signaling pathway. In conclusion, this work demonstrated that HOXC10 promoted growth and migration of melanoma by regulating Slug to activate the YAP/TAZ signaling pathway. Therefore, this study suggests that inhibition of HOXC10 has therapeutic potential in melanoma.

A new type of surgery for the treatment of bromhidrosis.

It is common to treat bromhidrosis by surgery, but postoperative complications such as subcutaneous exudate and subcutaneous hematoma can occur and lead to delayed healing of the wound and eventually lead to the formation of unattractive scars. In this study, we evaluated our new surgical treatment for bromhidrosis, which we believe improves prognosis over conventional surgery.The new procedure was performed on 22 patients with bromhidrosis. Our procedure is as follows. One centimeter-long incisions are made along the skin and cleaning of the subcutaneous apocrine glands using a special serrated scraping device is completed. Then, several 0.5 cm-long drainage holes are made according to the design of the Sudoku puzzle and 4 anchoring points identified to stabilize the oil gauze. Finally, the incisions were sutured and the wound covered with a bandage.Of 44 axillas, the bromhidrosis of 42 axillas was completely cured, and greatly reduced in 2 axillas. Local epidermal necrosis occurred in 5 axillas, but there was no full-thickness skin necrosis. Subcutaneous hematoma was not observed, and postoperative scarring was minimal.We found that our modified surgery can effectively reduce the occurrence of subcutaneous hematoma, avoid delayed healing of the wound, and minimize postoperative scarring.Level II, therapeutic study.

Severe Burn Injury Progression and Phasic Changes of Gene Expression in Mouse Model.

Patients with severe burns are susceptible to infectious complications including burn-site infections and sepsis. The purpose of this study was to explore the pathologic development of burn injury in a mouse model and to screen genes dysregulated at different time points on the basis of gene expression microarrays. Differential expression analysis identified a total 223 genes that related to only time progression independent of burn injury and 214 genes with aberrant expression due to burn injury. Weighted gene co-expression network analysis (WGCNA) of the 214 genes obtained seven gene modules which named as red, blue, turquoise, green, brown, yellow, and gray module, and the blue module was found to be significantly associated with severe burn injury progression, and in which several genes were previously reported being associated with inflammation and immune response, such as interleukin IL-6, IL-8, and IL-1b. Functional enrichment analysis indicated significant enrichment of biological processes that related to metabolism and catabolism, and pathways of proteasome, notch signaling and cell cycle. This result supports a phase progression of severe burn with gene expression changes and interpretation of biological processes in mouse.

Time series analysis of gene changes and processes after burn with human gene expression profiles.

Severe burns might be followed by severe infection associated with high mortality. In this study, we aimed to identify changes in genes and processes across time points after burn via analyzing time series gene expression profiles in burn patients and control from the Gene Expression Omnibus (GEO). Patients were classified into four groups according to time after burn and weighted gene co-expression network analysis (WGCNA) obtained three gene modules including magenta, yellow and greenyellow modules that significantly correlated positively with time after burn. We also identified four groups of differentially expressed genes (DEGs) in samples at 0-1d, 1-2d, 2-4d, and 4-7d after burn compared with controls. Functional enrichment analysis of those DEGs indicated significant enrichment of inflammatory/immune related processes throughout time points after burn, while, samples at later time points were also closely associated with cell activation regulation related processes. Short time series expression analysis of overlapping genes among the four lists of DEGs screened out two temporal gene expression profiles that exhibited decreasing and increasing expression trend across times after burn, and genes contained in those two profiles might be related to pathologic changes after severe burn.

Treatment of Ischemia-Reperfusion Injury of the Skin Flap Using Human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs) Transfected with "F-5" Gene.

BACKGROUND Recent studies have shown that skin flap transplantation technique plays an important role in surgical procedures. However, there are many problems in the process of skin flap transplantation surgeries, especially ischemia-reperfusion injury, which directly affects the survival rate of the skin flap and patient prognosis after surgeries. MATERIAL AND METHODS In this study, we used a new method of the "stem cells-gene" combination therapy. The "F-5" gene fragment of heat shock protein 90-α (Hsp90-α) was transfected into human umbilical cord mesenchymal stem cells (hUC-MSCs) by genetic engineering technique. RESULTS The synergistic effects of "F-5" gene and hUC-MSCs in the treatment of ischemia-reperfusion injury of the skin flap were confirmed by histochemical and immunohistochemical methods. CONCLUSIONS This study showed that the hUC-MSCs transfected with "F-5" gene can effectively improve the repair of ischemia-reperfusion injury.

The Current State of Nanoparticle-Induced Macrophage Polarization and Reprogramming Research.

Macrophages are vital regulators of the host defense in organisms. In response to different local microenvironments, resting macrophages (M0) can be polarized into different phenotypes, pro-inflammatory (M1) or anti-inflammatory (M2), and perform different roles in different physiological or pathological conditions. Polarized macrophages can also be further reprogrammed by reversing their phenotype according to the changed milieu. Macrophage polarization and reprogramming play essential roles in maintaining the steady state of the immune system and are involved in the processes of many diseases. As foreign substances, nanoparticles (NPs) mainly target macrophages after entering the body. NPs can perturb the polarization and reprogramming of macrophages, affect their immunological function and, therefore, affect the pathological process of disease. Optimally-designed NPs for the modulation of macrophage polarization and reprogramming might provide new solutions for treating diseases. Systematically investigating how NPs affect macrophage polarization is crucial for understanding the regulatory effects of NPs on immune cells in vivo. In this review, macrophage polarization by NPs is summarized and discussed.

Benefit of HSP90α intervention on ischemia-reperfusion injury of venous blood-congested flaps.

In order to decrease the incidence of flap necrosis after reconstructive surgeries, new approaches are required. In the present study, a model of venous congested flaps in rats was established to test the heat shock protein (HSP) 90α, 'F-5', protein as an intervention therapy to alleviate ischemia-reperfusion injury. A recombinant plasmid pET15b-F-5 carrying the HSP90α gene was constructed and the induced protein was purified from bacterial cell cultures. The rats in the study were divided into three different intervention groups: group A rats were treated with normal saline prior to flap establishment, group B rats were treated with HSP90α, 'F-5', protein prior to flap establishment, and group C rats were treated with the same 'F-5' protein after the surgical procedure. Additionally, the reperfusion time-points, ischemia for 6 or 8 h (5 rats each), were established in each group. After set periods of time, the flaps were observed for skin appearance, blood flow, survival rate and histological changes including neovascularization and re-epithelialization. The results showed that the flaps in the rats pre-treated with 'F-5' protein performed better than the flaps of rats in the other two groups: the blood flow was higher, flap survival rate was increased, inflammatory cell infiltration was decreased and angiogenesis increased, and new skin structure was better completed by the end of the experiment. The parameters examind were improved for all the groups when the ischemia time was 6 h instead of 8 h. In conclusion, HSP90α intervention prior to flap establishment was shown to be beneficial in the model of ischemia-reperfusion injury in venous-congested flaps.

Blocking TRAIL-DR5 signaling with soluble DR5 alleviates acute kidney injury in a severely burned mouse model.

Acute kidney injury (AKI) predicts high mortality in severely burned patients. Apoptosis plays a significant role during AKI; however, the apoptotic mechanisms underlying AKI induced by burn injury are not clear. Here, we report a critical role for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-Death receptor 5 (DR5) signaling in the pathogenesis of AKI. C57BL/6 male mice were subjected to full thickness scald burn. Apoptosis was significantly up-regulated in mouse kidney 24 h after the burn. Meanwhile, the TRAIL and DR5 expression levels were significantly increased in the kidney 24 h after the burn. Soluble DR5 treatment reduced apoptotic cell death and alleviated kidney injury induced by the burn through blocking the interaction of endogenous TRAIL with DR5. These results demonstrated that TRAIL plays a deleterious role in AKI pathogenesis induced by scald burns. Inhibition of TRAIL function in the kidney may represent a novel protective strategy to treat AKI in patients with burns.

Local transplant of human umbilical cord matrix stem cells improves skin flap survival in a mouse model.

A skin flap is a piece of skin that has its own blood supply, which is useful to repair large skin defects and deep wounds in plastic surgery. However, partial skin flap necrosis usually occurred. Bone marrow mesenchymal stem cells (BM MSCs) are effective in improving the ischemic flap survival, but their clinical application is restricted by their limited source. Human umbilical cord matrix stem (HUCMS) cells are easily isolated in a large number, compared to BM MSCs. In this study, we evaluated the therapeutic potential of HUCMS to improve the survival of ischemic skin flap. HUCMS cells were characterized with surface markers, and were labeled with 5-acetylene base-2 'deoxidizing uracil nucleoside (EdU) in vitro. Twenty male immunodeficient BALB/c mice with an epigastric flap were randomly divided into two groups. HUCMS cells or Dulbecco's Modified Eagle Medium (DMEM) were injected into the subcutaneous flap tissues. On the 7th postoperative day, flap survival, capillary density, levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), and EdU-positive cells in the skin flap were examined. Results showed that flap survival rate was higher in the HUCMS cell group (P < 0.05). Capillary density, VEGF level, and bFGF level were higher in the HUCMS cell group (P < 0.05). EdU-labeled HUCMS cells were mainly distributed in the subcutaneous flap tissues. These findings suggest that HUCMS cells can improve the survival of ischemic skin flap by promoting vascularization, which may be attributed to the increased expression of VEGF and bFGF.

[Role of lymphatics in bacterial translocation from intestine in burn rats].

OBJECTIVE: To investigate the role of lymphatics in bacterial translocation from intestine of rats with burn. METHODS: Escherichia coli (E. coli) labeled with chloromethylbenzamidodialkylcarbocyanine (CM-DIL) were prepared. Sixty adult male Wistar rats were randomly divided into scald group and sham injury group according to the envelope method, with 30 rats in each group. Rats in both groups were gavaged with 0.5 mL fluid containing CM-DIL-labeled E. coli. Rats in scald group were inflicted with 30% TBSA deep partial-thickness scald (verified by pathological section) and resuscitated with fluid. Rats in sham injury group were sham injured by bathing in 25 degrees C water for 10 s (verified by pathological section) and also received with fluid infusion. Mesenteric lymph node (MLN), liver, mesenteric lymph fluid (MLF), and liver vein blood (LVB) were harvested at post injury hour (PIH) 2, 24, and 72. Bacteria translocation was detected with fluorescent tracing technique and bacteria culture. The endotoxin content in above-mentioned four kinds of specimens was quantitatively determined with chromogenic substrate limulus amebocyte lysate. The carrying capacity of endotoxin in MLF and LVB was calculated. Data were processed with t test or one-way analysis of variance. RESULTS: (1) Living bacteria were in short-stick form, and they were seen moving in single or in doubles or triples in sample fluid. Dead bacteria were in irregular aggregates. Labeled bacteria in small amount were detected in sham injury group, their number peaked at PIH 24. A large amount of labeled bacteria were detected in scald group at PIH 2, which peaked at PIH 24 and decreased at PIH 72. The largest amount of labeled bacteria were found in MLN in scald group as compared to those in the other samples, and the number peaked at PIH 24 [(5872 +/- 1976) x 10(3) CFU/g], which was obviously higher than that [(216 +/- 110) x 10(3) CFU/g, t = 30.129, P = 0.000] in sham injury group. The number of bacteria decreased at PIH 72, but it was still significantly different from that in sham injury group ( t = 4.323, P = 0.000). The number of bacteria in LVB was the smallest. (2) 29 (24.2%) samples out of the 120 samples in sham injury group were positive for bacteria. 72 (60.0%) samples out of the 120 samples in scald group were positive for bacteria. No alive bacterium was detected at any time point in LVB sample in both group; the other three samples were detected with alive bacteria since PIH 2. There were more alive bacteria detected in MLN and liver as compared with the other two kinds of samples in scald group. The amount of bacteria in MLN, liver, and MLF in scald group were higher than those in sham injury group (with t value respectively 4.353, 4.354, 4.965, P values all equal to 0.000). (3) The endotoxin level in each kind of sample at each time point was obviously higher in scald group than that in sham injury group, and it peaked at PIH 2 in liver and MLF. The difference of endotoxin level among 4 kinds of samples in scald group at PIH 2 was statistically significant ( F = 258.47, P = 0.000), and the endotoxin level was higher in liver, MLN, and MLF. They were obviously higher than those in sham injury group (with t value respectively 43.378, 43.123, 22.423, P values all equal to 0.000). The endotoxin level in MLF was 9 times of that in LVB. (4) The carrying capacity of endotoxin in LVB and MLF at each time point in scald group was higher than that in sham injury group. CONCLUSIONS: CM-DIL marked bacteria can reflect the microbial translocation condition. The lymphatic route is an important pathway for bacteria translocation.

Lymph invasion route of bacteria and endotoxin and CD4+/CD8+ T cells ratio changes of draining lymph node of burn infection wound.

The aim of this study was to determine the lymphatic invasion route of bacteria and endotoxin of burn wounds infected by Pseudomonas aeruginosa and moreover, the effect of P. aeruginosa infection of the burn wound on the draining lymph node and lymph fluid. Male Wistar rats were subjected to unilateral hind limb burn+wound infected by P. aeruginosa (infection limbs group) and contralateral hind limb burn alone (burn limbs group). On hours 6, 24 and 72 after infection, rats were killed, the common iliac lymph nodes (CILN) was collected for the culture of P. aeruginosa. Lymph fluid in the efferent lymph trunk of CILN was collected for the measurement of endotoxin by the Limulus Amebocyte Lysate test. Lymph fluid tumor necrosis factor-alpha (TNF-alpha) concentration was measured by enzyme-linked immunosorbent assay (ELISA). The CD4+/CD8+ T cells ratio of CILN was subjected to flow cytometry analysis. The results showed bacteria invasion incidence, endotoxin and TNF-alpha concentrations were significantly higher in the infection limb group when compared to the burn limb group (P<0.01). The CD4+/CD8+ T cell ratio was significantly lower on post-burn wound infection hours 72 (P<0.05). This study provides evidence that bacteria and endotoxin of burn wound infected by P. aeruginosa invade draining lymph node and lymph fluid. P. aeruginosa infection of the burn wound can increase TNF-alpha level of the draining lymph fluid and decrease CD4+/CD8+ T cells ratio of the draining lymph node.

[Expression of tumor necrosis factor related apoptosis inducing ligand and its receptor in thymus during early post-burn stage in rat with severe burns].

OBJECTIVE: To investigate the effect of tumor necrosis factor related apoptosis inducing ligand (TRAIL) and its receptor on apoptosis in thymus during early post-burn stage in rat with severe burns. METHODS: Fifty Wistar rats were randomly divided into sham scald group (SS, n = 10) and burn group (n = 40). The apoptosis in thymus in rats was detected with annexin V/FITC-PI double staining at 4, 12, 24, 48 post-burn hours (PBH). The expression of TRAIL death receptor DR5, DR4 and its decoy receptor DcR1, DcR2 in thymus were detected by RT-PCR and Western blot at above time-points. RESULTS: Compared with that in SS group (6.7 +/- 0.8)%, the apoptosis in the thymus in burn group started to increase at 4 PBH [(17.1 +/- 0.4)%], peaked at 12 PBH [(25.2 +/- 1.1)%], and it was still evidently higher than that in SS group at 48 PBH (P < 0.05). There was no obvious difference in the apoptosis rate in rats in burn group among all the time-points. The expression of DR5 in burn group at each time-points was significantly higher than those in SS group, while that of DcR2 shown an opposite tendency (P < 0.05). The expression of DR4, DcR1 was similar in both groups. CONCLUSION: The marked increase in apoptosis rate in rat thymus at early post-burn stage, and the significant change in the expression of DR5 and DcR2 show that TRAIL pathway may participate in apoptosis.

[The changes in the tumor necrosis factor alpha, interleukin-6 and interleukin-8 levels in the lymph and of the dynamics of the lymphokines during shock stage of rats with major burns].

OBJECTIVE: To study the dynamic changes in the lymphokines and the changes in tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-8 (IL-8) levels in the lymph during shock stage of rats with major burns. METHODS: Forty-two male adult Wistar rats were randomly divided into burn resuscitation group (A, n = 18), burn non-resuscitation (B, n = 18) and the control (C, n = 6) groups. The TNF-alpha, IL-6 and IL-8 levels in the lymph were determined with radioimmunoassay at 6, 24, 48 postburn hours (PBH). The lymphokines in the mesenteric lymphatic vessels was observed at 6, 24 and 48 PBH with inverted microscopy and digital image processing, and the contraction frequency of the lymphatic was calculated. The lymph was collected by cannulation of the chylous cistern, and its speed of flow was calculated. RESULTS: The lymphatic contents of TNF-alpha and IL-6 in both A and B groups began to increase at 6PBH, reaching the peak values at 24 PBH (TNF-alpha in A and B groups were 1.61 +/- 0.27 ug/L and 1.86 +/- 0.34 ug/L, respectively; IL-6 in A and B groups were 398 +/- 67 ng/L and 572 +/- 97 ng/L, respectively), and they were significantly higher than those in C group at each time points (P < 0.01), meanwhile there was also obvious difference in them between A and B groups (P < 0.01). The lymphatic contents of IL-8 in A and B groups began to increase at 24 PBH, and continued to increase till 48PBH (540.29 +/- 0.32 ng/L in A group, 863.48 +/- 105.16 ng/L in B group), which were evidently higher than those in C group (P < 0.01). There was significant difference in IL-8 contents between A and B groups (P < 0.01). The contraction frequency of the mesenteric lymphatic vessels in A and B groups were decreased, especially so at 24 PBH (P < 0.01). The speed of lymphatic flow in A and B groups was increased at each time points (P < 0.01). The central chylous vessels in the villi of the small intestine were extremely dilated as seen under microscope. CONCLUSION: After burn injury, the lymphatic vessels dilated, with its motility decreased and speed of flow increased, and the contents of TNF-alpha, IL-6 and IL-8 in lymph were increased during the shock stage of burn rats. Fluid resuscitation could improve the lymph circulation.

[Expression and significance of TRAIL receptors in fibroblasts of hypertrophic scar].

OBJECTIVE: To detect the expression of TRAIL receptors in fibroblasts of hypertrophic scar in the proliferative stage and explore its significance. METHODS: 30 samples of hypertrophic scar were taken from 30 burn cases in the proliferative stage. 30 samples of normal skin were taken as the control. The expressions of TRAIL receptors in the fibroblasts of hypertrophic burn scar and the normal skin were assayed by semiquantitative RT-PCR and flowcytometry. RESULTS: The expression level of DR5 in the fibroblasts of hypertrophic burn scar is much lower than the control (P < 0.05); the expression level of DcR1 in the fibroblasts of hypertrophic burn scar is much higher than the control (P < 0.05). CONCLUSIONS: The down-regulated DR5 expression and elevated DcR1 expressions in the fibroblasts of hypertrophic burn scar may attribute to the apoptosis change induced by TRAIL and explain the apoptosis differences between the fibroblasts of hypertrophic scar and normal skin to a certain extent.