Angiogenesis in Wounds Treated by Microdeformational Wound Therapy.

BACKGROUND:: Mechanical forces play an important role in tissue neovascularization and are a constituent part of modern wound therapies. The mechanisms by which vacuum assisted closure (VAC) modulates wound angiogenesis are still largely unknown. OBJECTIVE:: To investigate how VAC treatment affects wound hypoxia and related profiles of angiogenic factors as well as to identify the anatomical characteristics of the resultant, newly formed vessels. METHODS:: Wound neovascularization was evaluated by morphometric analysis of CD31-stained wound cross-sections as well as by corrosion casting analysis. Wound hypoxia and mRNA expression of HIF-1α and associated angiogenic factors were evaluated by pimonidazole hydrochloride staining and quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Vascular endothelial growth factor (VEGF) protein levels were determined by western blot analysis. RESULTS:: VAC-treated wounds were characterized by the formation of elongated vessels aligned in parallel and consistent with physiologically function, compared to occlusive dressing control wounds that showed formation of tortuous, disoriented vessels. Moreover, VAC-treated wounds displayed a well-oxygenated wound bed, with hypoxia limited to the direct proximity of the VAC-foam interface, where higher VEGF levels were found. By contrast, occlusive dressing control wounds showed generalized hypoxia, with associated accumulation of HIF-1α and related angiogenic factors. CONCLUSIONS:: The combination of established gradients of hypoxia and VEGF expression along with mechanical forces exerted by VAC therapy was associated with the formation of more physiological blood vessels compared to occlusive dressing control wounds. These morphological changes are likely a necessary condition for better wound healing.

Effects of triamcinolone acetonide on vessels of the posterior segment of the eye.

PURPOSE: This study investigates the effects of triamcinolone acetonide (TA) on retinal endothelial cells in vitro and explores the potential vascular toxic effect of TA injected into the vitreous cavity of rats in vivo. METHODS: Subconfluent endothelial cells were treated with either 0.1 mg/ml or 1 mg/ml TA in 1% ethanol. Control cells were either untreated or exposed to 1% ethanol. Cell viability was evaluated at 24 h, 72 h, and five days using the tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5 phenyltetrazolium bromide test (MTT) and lactate dehydrogenase (LDH) assays. Cell proliferation was evaluated by 5-bromo-2-deoxyuridine (BrdU) test. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL assay), annexin-binding, and caspase 3 activation. Caspase-independent cell deaths were investigated by immunohistochemistry using antibodies against apoptosis inducing factor (AIF), cytochrome C, microtubule-associated protein (MAP)-light chain 3 (MAP-LC3), and Leukocyte Elastase Inhibitor/Leukocyte Elastase Inhibitor-derived DNase II (LEI/L-DNase II). In vivo, semithin and ultrathin structure analysis and vascular casts were performed to examine TA-induced changes of the choroidal vasculature. In addition, outer segments phagocytosis assay on primary retinal pigment epithelium (RPE) cells was performed to assess cyclooxygenase (COX-2) and vascular endothelial growth factor (VEGF) mRNAs upregulation with or without TA. RESULTS: The inhibitory effect of TA on cell proliferation could not explain the significant reduction in cell viability. Indeed, TA induced a time-dependent reduction of bovine retinal endothelial cells viability. Annexin-binding positive cells were observed. Cytochrome C was not released from mitochondria. L-DNase II was found translocated to the nucleus, meaning that LEI was changed into L-DNase II. AIF was found nuclearized in some cells. LC3 labeling showed the absence of autophagic vesicles. No autophagy or caspase dependent apoptosis was identified. At 1 mg/ml TA induced necrosis while exposure to lower concentrations for 3 to 5 days induced caspase independent apoptosis involving AIF and LEI/L-DNase II. In vivo, semithin and ultrathin structure analysis and vascular casts revealed that TA mostly affected the choroidal vasculature with a reduction of choroidal thickness and increased the avascular areas of the choriocapillaries. Experiments performed on primary RPE cells showed that TA downregulates the basal expression of COX-2 and VEGF and inhibits the outer segments (OS)-dependent COX-2 induction but not the OS-dependent VEGF induction. CONCLUSIONS: This study demonstrates for the first time that glucocorticoids exert direct toxic effect on endothelial cells through caspase-independent cell death mechanisms. The choroidal changes observed after TA intravitreous injection may have important implications regarding the safety profile of TA use in human eyes.

A morphometric study of mechanotransductively induced dermal neovascularization.

BACKGROUND:: Mechanical stretch has been shown to induce vascular remodeling and increase vessel density, but the pathophysiologic mechanisms and the morphologic changes induced by tensile forces to dermal vessels are poorly understood. METHODS:: A custom computer-controlled stretch device was designed and applied to the backs of C57BL/6 mice (n = 38). Dermal and vascular remodeling was studied over a 7-day period. Corrosion casting and three-dimensional scanning electron microscopy and CD31 staining were performed to analyze microvessel morphology. Hypoxia was assessed by immunohistochemistry. Western blot analysis of vascular endothelial growth factor (VEGF) and mRNA expression of VEGF receptors was performed. RESULTS:: Skin stretching was associated with increased angiogenesis as demonstrated by CD31 staining and vessel corrosion casting where intervascular distance and vessel diameter were decreased (p < 0.01). Immediately after stretching, VEGF dimers were increased. Messenger RNA expression of VEGF receptor 1, VEGF receptor 2, neuropilin 1, and neuropilin 2 was increased starting as early as 2 hours after stretching. Highly proliferating epidermal cells induced epidermal hypoxia starting at day 3 (p < 0.01). CONCLUSIONS:: Identification of significant hypoxic cells occurred after identification of neovessels, suggesting an alternative mechanism. Increased expression of angiogenic receptors and stabilization of VEGF dimers may be involved in a mechanotransductive, prehypoxic induction of neovascularization.

Impact of platelet rich plasma and adipose stem cells on lymphangiogenesis in a murine tail lymphedema model.

BACKGROUND: Lymphedema is an underdiagnosed pathology which in industrialized countries mainly affects cancer patients that underwent lymph node dissection and/or radiation. Currently no effective therapy is available so that patients' life quality is compromised by swellings of the concerned body region. This unfortunate condition is associated with body imbalance and subsequent osteochondral deformations and impaired function as well as with an increased risk of potentially life threatening soft tissue infections. METHODS: The effects of PRP and ASC on angiogenesis (anti-CD31 staining), microcirculation (Laser Doppler Imaging), lymphangiogenesis (anti-LYVE1 staining), microvascular architecture (corrosion casting) and wound healing (digital planimetry) are studied in a murine tail lymphedema model. RESULTS: Wounds treated by PRP and ASC healed faster and showed a significantly increased epithelialization mainly from the proximal wound margin. The application of PRP induced a significantly increased lymphangiogenesis while the application of ASC did not induce any significant change in this regard. CONCLUSIONS: PRP and ASC affect lymphangiogenesis and lymphedema development and might represent a promising approach to improve regeneration of lymphatic vessels, restore disrupted lymphatic circulation and treat or prevent lymphedema alone or in combination with currently available lymphedema therapies.

Testicular ultrastructure and morphology of the seminal pathway in Prochilodus scrofa

The seminiferous tubules of Prochilodus scrofa present a coiled morphological arrangement with intertubular anastomoses and unrestricted spermatogonial distribution. The structural pattern of the seminiferous tubules is cystic, with cysts formed by cytoplasmic prolongations of Sertoli cells. Inside the cysts are observed different types of germ cells. The seminiferous tubules open individually on the ventral surface of the main testicular duct present in each testis. Each main testicular duct prolongs as a spermatic duct, fusing with the spermatic duct of the opposite side to form the common spermatic duct which opens into the urogenital papilla. The mature sperm cysts break and extravasate their content into the lumen of the seminiferous tubules from which the seminal fluid and the spermatozoa penetrate the main testicular duct, the spermatic duct and the common spermatic duct for semen ejaculation.

Spatial and functional relationships between air conduits and blood capillaries in the pulmonary gas exchange tissue of adult and developing chickens

The documented data regarding the three-dimensional structure of the air capillaries (ACs), the ultimate sites of gas exchange in the avian lung is contradictory. Further, the mode of gas exchange, described as cross-current has not been clearly elucidated. We studied the temporal and spatial arrangement of the terminal air conduits of the chicken lung and their relationship with the blood capillaries (BCs) in embryos as well as the definitive architecture in adults. Several visualization techniques that included corrosion casting, light microscopy as well as scanning and transmission electron microscopy were used. Two to six infundibulae extend from each atrium and give rise to numerous ACs that spread centrifugally. Majority of the ACs are tubular structures that give off branches, which anastomose with their neighboring cognates. Some ACs have globular shapes and a few are blind-ending tapering tubes. During inauguration, the luminal aspects of the ACs are characterized by numerous microvillus-like microplicae, which are formed during the complex processes of cell attenuation and canalization of the ACs. The parabronchial exchange BCs, initially inaugurated as disorganized meshworks, are reoriented via pillar formation to lie predominantly orthogonal to the long axes of the ACs. The remodeling of the retiform meshworks by intussusceptive angiogenesis essentially accomplishes a cross-current system at the gas exchange interface in the adults, where BCs form ring-like patterns around the ACs, thus establishing a cross-current system. Our findings clarify the mode of gas exchange in the parabronchial mantle and illuminate the basis for the functional efficiency of the avian lung.

Sprouting and intussusceptive angiogenesis in postpneumonectomy lung growth: mechanisms of alveolar neovascularization

In most rodents and some other mammals, the removal of one lung results in compensatory growth associated with dramatic angiogenesis and complete restoration of lung capacity. One pivotal mechanism in neoalveolarization is neovascularization, because without angiogenesis new alveoli can not be formed. The aim of this study is to image and analyze three-dimensionally the different patterns of neovascularization seen following pneumonectomy in mice on a sub-micron-scale. C57/BL6 mice underwent a left-sided pneumonectomy. Lungs were harvested at various timepoints after pneumonectomy. Volume analysis by microCT revealed a striking increase of 143 percent in the cardiac lobe 14 days after pneumonectomy. Analysis of microvascular corrosion casting demonstrated spatially heterogenous vascular densitities which were in line with the perivascular and subpleural compensatory growth pattern observed in anti-PCNA-stained lung sections. Within these regions an expansion of the vascular plexus with increased pillar formations and sprouting angiogenesis, originating both from pre-existing bronchial and pulmonary vessels was observed. Also, type II pneumocytes and alveolar macrophages were seen to participate actively in alveolar neo-angiogenesis after pneumonectomy. 3D-visualizations obtained by high-resolution synchrotron radiation X-ray tomographic microscopy showed the appearance of double-layered vessels and bud-like alveolar baskets as have already been described in normal lung development. Scanning electron microscopy data of microvascular architecture also revealed a replication of perialveolar vessel networks through septum formation as already seen in developmental alveolarization. In addition, the appearance of pillar formations and duplications on alveolar entrance ring vessels in mature alveoli are indicative of vascular remodeling. These findings indicate that sprouting and intussusceptive angiogenesis are pivotal mechanisms in adult lung alveolarization after pneumonectomy. Various forms of developmental neoalveolarization may also be considered to contribute in compensatory lung regeneration.

Impact of platelet rich plasma and adipose stem cells on lymphangiogenesis in a murine tail lymphedema model.

BACKGROUND Lymphedema is an underdiagnosed pathology which in industrialized countries mainly affects cancer patients that underwent lymph node dissection and/or radiation. Currently no effective therapy is available so that patients' life quality is compromised by swellings of the concerned body region. This unfortunate condition is associated with body imbalance and subsequent osteochondral deformations and impaired function as well as with an increased risk of potentially life threatening soft tissue infections. METHODS The effects of PRP and ASC on angiogenesis (anti-CD31 staining), microcirculation (Laser Doppler Imaging), lymphangiogenesis (anti-LYVE1 staining), microvascular architecture (corrosion casting) and wound healing (digital planimetry) are studied in a murine tail lymphedema model. RESULTS Wounds treated by PRP and ASC healed faster and showed a significantly increased epithelialization mainly from the proximal wound margin. The application of PRP induced a significantly increased lymphangiogenesis while the application of ASC did not induce any significant change in this regard. CONCLUSIONS PRP and ASC affect lymphangiogenesis and lymphedema development and might represent a promising approach to improve regeneration of lymphatic vessels, restore disrupted lymphatic circulation and treat or prevent lymphedema alone or in combination with currently available lymphedema therapies.

Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice

A physiological examination of mice harboring a null allele at the aryl hydrocarbon (Ah) locus revealed that the encoded aryl hydrocarbon receptor plays a role in the resolution of fetal vascular structures during development. Although the aryl hydrocarbon receptor is more commonly studied for its role in regulating xenobiotic metabolism and dioxin toxicity, a developmental role of this protein is supported by the observation that Ah null mice display smaller livers, reduced fecundity, and decreased body weights. Upon investigating the liver phenotype, we found that the decrease in liver size is directly related to a reduction in hepatocyte size. We also found that smaller hepatocyte size is the result of massive portosystemic shunting in null animals. Colloidal carbon uptake and microsphere perfusion studies indicated that 56% of portal blood flow bypasses the liver sinusoids. Latex corrosion casts and angiography demonstrated that shunting is consistent with the existence of a patent ductus venosus in adult animals. Importantly, fetal vascular structures were also observed at other sites. Intravital microscopy demonstrated an immature sinusoidal architecture in the liver and persistent hyaloid arteries in the eyes of adult Ah null mice, whereas corrosion casting experiments described aberrations in kidney vascular patterns.

Microstructural characteristics and corrosion behavior of a super duplex stainless steel casting

The machining of super duplex stainless steel castings is usually complicated by the difficulty involved in maintaining the dimensional tolerances required for given applications. Internal stresses originating from the solidification process and from subsequent heat treatments reach levels that exceed the material`s yield strength, promoting plastic strain. Stress relief heat treatments at 520 degrees C for 2 h are an interesting option to solve this problem, but because these materials present a thermodynamically metastable condition, a few precautions should be taken. The main objective of this work was to demonstrate that, after solution annealing at 1130 degrees C and water quenching, stress relief at 520 degrees C for 2 h did not alter the duplex microstructure or impair the pitting corrosion resistance of ASTM A890/A890M Grade 6A steel. This finding was confirmed by microstructural characterization techniques, including light optical and scanning electron microscopy, and X-ray diffraction. Corrosion potential measurements in synthetic sea water containing 20,000 ppm of chloride ions were also conducted at three temperatures: 5 degrees C, 25 degrees C and 60 degrees C. (c) 2008 Elsevier Inc. All rights reserved.

Influence of microstructure on the corrosion of diecast AZ91D

The corrosion of die cast AZ91D was studied and related to its microstructure. For comparison and to more fully understand the behaviour of die cast AZ91D, corrosion studies and microstructural examinations were also carried out using slowly solidified high purity AZ91, Mg-2%Al, Mg-9%Al, low purity magnesium and high purity magnesium. Corrosion was studied in 1N NaCl at pH 11 by (1) observing the corrosion morphology, (2) measuring electrochemical polarisation curves and (3) simultaneously measuring both the hydrogen evolution rate and the magnesium dissolution rate. The skin of die cast AZ91D showed better corrosion resistance than the interior. This is attributed to a combination of(1) a higher volume fraction of the beta phase, (2) a more continuous beta phase distribution around finer alpha grains, and (3) lower porosity in the skin layer than in the interior of the die casting. This study showed that the casting method can influence the corrosion performance by its influence on the alloy microstructure. (C) 1999 Elsevier Science Ltd. All rights reserved.

In vitro cytotoxicity of dental alloys and cpTi obtained by casting

Background: This study aimed to compare the cytotoxicity of base-metal dental alloys and to evaluate if the casting method could influence their cytotoxicity. Methods: Disks of base-metal dental alloys were cast by two methods: plasma, under argon atmosphere, injected by vacuum-pressure; and oxygen-gas flame, injected by centrifugation, except Ti-6Al-4V and commercially pure titanium (cpTi), cast only by plasma. SCC9 cells were cultured in culture media D-MEM/Ham`s F12 supplemented, at 37 degrees C in a humidified atmosphere of 5% carbon dioxide and 95% air, on the previously prepared disks. At subconfluence in wells without disks (control), cell number and viability were evaluated. Results: In plasma method, cpTi and Ti-6Al-4V were similar to control and presented higher number of cells than all other alloys, followed by Ni-Cr. In oxygen-gas name method, all alloys presented fewer cells than control. Ni-Cr presented more cells than any other alloy, followed by Co-Cr-Mo-W which presented more cells than Ni-Cr-Ti, Co-Cr-Mo, and Ni-Cr-Be. There were no significant differences between casting methods related to cell number. Cell viability was not affected by either chemical composition or casting methods. Conclusion: cpTi and Ti-6Al-4V were not cytotoxic while Ni-Cr-Be was the most cytotoxic among tested alloys. The casting method did not affect cytotoxicity of the alloys. (c) 2007 Wiley Periodicals, Inc.

Estudio de los procesos de corrosion de una aleacion metalica de cobre usada en odontologia

Corrosion phenomena in a dental copper-based alloy are experimentally studied using electrochemical techniques. Two heating sources, torch and induction, were used in the casting experiments. In the corrosion essays, the optimum casting cycle and the polarization curves were obtained. It was found that the heating sources have little influence on the corrosion processes of the metallic alloys studied.

Study of corrosion resistance of laser welded Au-Pd-Ag-In alloy using electrochemical.

The aim of this work was to evaluate the corrosion resistance of AuPdAgIn alloy, submitted to laser beam welding, in 0.9% NaCl solution, using electrochemical techniques. Measures of the open circuit potential (OCP) versus time were applied to electrochemical experiments, as well as potentiodynamic direct scanning (PDS) and electrochemical impedance spectroscopy (EIS) on AuPdAgIn alloy, submitted to laser beam welding in 0.9% NaCl solution. Some differences observed in the microstructure can explain the results obtained for corrosion potential, Ecorr, and corrosion resistance, Rp. EIS spectra have been characterized by distorted capacitive components, presenting linear impedance at low frequencies, including a non-uniform diffusion. The area of the laser weld presented corrosion potential slightly superior when compared to the one of the base metal. The impedance results suggest the best resistant corrosion behavior for laser weld than base metal region. This welding process is a promising alternative to dental prostheses casting.

Corrosion behaviour of aluminium syntactic functionally graded composites

Syntactic Functionally Graded Metal Matrix Composites (SFGMMC) are a type of composites reinforced by microballoons exhibiting a graded reinforcement distribution. These materials constitute a promising new generation of lightweight structural materials for aerospace, marine and shielding/insulation applications. In this work, A356 alloy reinforced with silica-alumina microballoons (SiO2-Al2O3) was processed by casting techniques. The influence of the microballoon distribution gradient on the corrosion behaviour of the composite was investigated by potentiodynamic polarisation and Electrochemical Impedance Spectroscopy (EIS). Composite surfaces were analysed before and after testing by Optical Microscopy (OM) and Scanning Electron Microscopy (SEM) to determine the influence of microstructural changes.