Hybrid Scaffolds Built From PET and Collagen as a Model For Vascular Graft Architecture

A hybrid material with excellent mechanical and biological properties is produced by electrospinning a co-solution of PET and collagen. The fibers are mapped using SEM, confocal Raman microscopy and collagenase digestion assays. Fibers of different compositions and morphologies are intermingled within the same membrane, resulting in a heterogeneous scaffold. The collagen distribution and exposure are found to depend on the PET/collagen ratio. The materials are chemically and mechanically characterized and biologically tested with fibroblasts (3T3-L1) and a HUVEC culture in vitro. All of the hybrid scaffolds show better cell attachment and proliferation than PET. These materials are potential candidates to be used as vascular grafts.

On the temperature stability of extracellular hemoglobin of Glossoscolex paulistus, at different oxidation states: SAXS and DLS studies

Glossoscolex paulistus hemoglobin (HbGp) was studied by dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). DLS melting curves were measured for met-HbGp at different concentrations. SAXS temperature studies were performed for oxy-, cyanomet- and met-HbGp forms, at several pH values. At pH 5.0 and 6.0, the scattering curves are identical from 20 to 60 degrees C, and R-g is 108 angstrom, independent of the oxidation form. At pH 7.0, protein denaturation and aggregation occurs above 55 degrees C and 60 degrees C, for oxy and met-HbGp, respectively. Cyanomet-HbGp, at pH 7.0, is stable up to 60 degrees C. At alkaline pH (8.0-9.0) and higher temperature, an irreversible dissociation process is observed, with a decrease of R-g, D-max and I(0). Analysis by p(r), obtained from GNOM, and OLIGOMER, was used to fit the SAXS experimental scattering curves by a combination of theoretical curves obtained for HbLt fragments from the crystal structure. Our results show clearly the increasing contribution of smaller molecular weight fragments, as a function of increasing pH and temperature, as well as, the order of thermal stabilities: cyanomet-> oxy- > met-HbGp. (C) 2012 Elsevier B.V. All rights reserved.

Extracellular matrix composition in COPD

Extracellular matrix (ECM) composition has an important role in determining airway structure. We postulated that ECM lung composition of chronic obstructive pulmonary disease (COPD) patients differs from that observed in smoking and nonsmoking subjects without airflow obstruction. We determined the fractional areas of elastic fibres, type-I, -III and -IV collagen, versican, decorin, biglycan, lumican, fibronectin and tenascin in different compartments of the large and small airways and lung parenchyma in 26 COPD patients, 26 smokers without COPD and 16 nonsmoking control subjects. The fractional area of elastic fibres was higher in non-obstructed smokers than in COPD and nonsmoking controls, in all lung compartments. Type-I collagen fractional area was lower in the large and small airways of COPD patients and in the small airways of non-obstructed smokers than in nonsmokers. Compared with nonsmokers, COPD patients had lower versican fractional area in the parenchyma, higher fibronectin fractional area in small airways and higher tenascin fractional area in large and small airways compartments. In COPD patients, significant correlations were found between elastic fibres and fibronectin and lung function parameters. Alterations of the major ECM components are widespread in all lung compartments of patients with COPD and may contribute to persistent airflow obstruction.

In vitro influence of the extracellular matrix in myoepithelial cells stimulated by malignant conditioned medium

In order to investigate the role of myoepithelial cell and tumor microenvironment in salivary gland neoplasma, we have performed a study towards the effect of different extracellular matrix proteins (basement membrane matrix, type I collagen and fibronectin) on morphology and differentiation of benign myoepithelial cells from pleomorphic adenoma cultured with malignant cell culture medium from squamous cell carcinoma. We have also analyzed the expression of alpha-smooth muscle actin (alpha-SMA) and FGF-2 by immunofluorescence and qPCR. Our immunofluorescence results, supported by qPCR analysis, demonstrated that alpha-SMA and FGF-2 were upregulated in the benign myoepithelial cells from pleomorphic adenoma in all studied conditions on fibronectin substratum. However, the myoepithelial cells on fibronectin substratum did not alter their morphology under malignant conditioned medium stimulation and exhibited a stellate morphology and, occasionally focal adhesions with the substratum. In summary, our data demonstrated that the extracellular matrix exerts an important role in the morphology of the benign myoepithelial cells by the presence of focal adhesions and also inducing increase FGF-2 and alpha-SMA expression by these cells, especially in the fibronectin substratum. (C) 2011 Elsevier Ltd. All rights reserved.

The Extracellular Matrix of the Lateral Pharyngeal Wall in Obstructive Sleep Apnea

Study Objectives: To compare the components of the extracellular matrix in the lateral pharyngeal muscular wall in patients with and without obstructive sleep apnea (OSA). This may help to explain the origin of the increased collapsibility of the pharynx in patients with OSA. Design: Specimens from the superior pharyngeal constrictor muscle, obtained during pharyngeal surgeries, were evaluated using histochemical and immunohistochemical analyses to determine the fractional area of collagen types I and II, elastic fibers, versican, fibronectin, and matrix metalloproteinases 1 and 2 in the endomysium. Setting: Academic tertiary center. Patiens: A total of 51 nonobese adult patients, divided into 38 patients with OSA and 13 nonsnoring control subjects without OSA. Interventions: Postintervention study performed on tissues from patients after elective surgery. Measurements and Results: Pharyngeal muscles of patients with OSA had significantly more collagen type I than pharyngeal muscles in control subjects. Collagen type I was correlated positively and independently with age. The other tested components of the extracellular matrix did not differ significantly between groups. In a logistic regression, an additive effect of both the increase of collagen type I and the increase in age with the presence of OSA was observed (odds ratio (OR), 2.06; 95% confidence interval (CI), 1.17-3.63), when compared with the effect of increased age alone (OR, 1.11; 95% CI, 1.03-1.20). Conclusion: Collagen type I in the superior pharyngeal constrictor muscle was more prevalent in patients with OSA and also increased with age. It was hypothesized that this increase could delay contractile-relaxant responses in the superior pharyngeal constrictor muscle at the expiratory-inspiratory phase transition, thus increasing pharyngeal collapsibility.

Characterization of a thermostable extracellular tannase produced under submerged fermentation by Aspergillus ochraceus

Background: Tannases are enzymes that may be used in different industrial sectors as, for example, food and pharmaceutical. They are obtained mainly from microorganisms, as filamentous fungi. However, the diversity of fungi stays poorly explored for tannase production. In this article, Aspergillus ochraceus is presented as a new source of tannase with interesting features for biotechnological applications. Results: Extracellular tannase production was induced when the fungus was cultured in Khanna medium with tannic acid as carbon source. The extracellular tannase was purified 9-fold with 2% recovery and a single band corresponding to 85 kDa was observed in SDS-PAGE. The native apparent molecular mass was estimated as 112 kDa. Optima of temperature and pH were 40 degrees C and 5.0, respectively. The enzyme was fully stable from 40 degrees C to 60 degrees C during 1 hr. The activity was enhanced by Mn2+ (33-39%) and NH4+ (15%). The purified tannase hydrolyzed tannic acid and methyl gallate with Km of 0.76 mM and 0.72 mM, respectively, and Vmax of 0.92 U/mg protein and 0.68 U/mg protein, respectively. The analysis of a partial sequence of the tannase encoding gene showed an open read frame of 567 bp and a sequence of 199 amino acids were predicted. TLC analysis revealed the presence of gallic acid as a tannic acid hydrolysis product. Conclusion: The extracellular tannase produced by A. ochraceus showed distinctive characteristics such as monomeric structure and activation by Mn2+, suggesting a new kind of fungal tannases with biotechnological potential. Further, it was the first time that a partial gene sequence for A. ochraceus tannase was described.

Glycosaminoglycans modulate C6 glioma cell adhesion to extracellular matrix components and alter cell proliferation and cell migration

Abstract Background Adhesion to extracellular matrix (ECM) components has been implicated in the proliferative and invasive properties of tumor cells. We investigated the ability of C6 glioma cells to attach to ECM components in vitro and described the regulatory role of glycosaminoglycans (GAGs) on their adhesion to the substrate, proliferation and migration. Results ECM proteins (type IV collagen, laminin and fibronectin) stimulate rat C6 glioma cell line adhesion in vitro, in a dose-dependent manner. The higher adhesion values were achieved with type IV collagen. Exogenous heparin or chondroitin sulfate impaired, in a dose-dependent manner the attachment of C6 glioma cell line to laminin and fibronectin, but not to type IV collagen. Dextran sulfate did not affect C6 adhesion to any ECM protein analyzed, indicating a specific role of GAGs in mediating glioma adhesion to laminin and fibronectin. GAGs and dextran sulfate did not induce C6 glioma detachment from any tested substrate suggesting specific effect in the initial step of cell adhesion. Furthermore, heparin and chondroitin sulfate impaired C6 cells proliferation on fibronectin, but not on type IV collagen or laminin. In contrast, both GAGs stimulate the glioma migration on laminin without effect on type IV collagen or fibronectin. Conclusion The results suggest that GAGs and proteoglycans regulate glioma cell adhesion to ECM proteins in specific manner leading to cell proliferation or cell migration, according to the ECM composition, thus modulating tumor cell properties.

Effects of myenteric denervation on extracellular matrix fibers and mast cell distribution in normal stomach and gastric lesions

Abstract Background In this study the effect of myenteric denervation induced by benzalconium chloride (BAC) on distribution of fibrillar components of extracellular matrix (ECM) and inflammatory cells was investigated in gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Rats were divided in four experimental groups: non-denervated (I) and denervated stomach (II) without MNNG treatment; non-denervated (III) and denervated stomachs (IV) treated with MNNG. For histopathological, histochemical and stereological analysis, sections of gastric fragments were stained with Hematoxylin-Eosin, Picrosirius-Hematoxylin, Gomori reticulin, Weigert's Resorcin-Fuchsin, Toluidine Blue and Alcian-Blue/Safranin (AB-SAF). Results BAC denervation causes an increase in the frequency of reticular and elastic fibers in the denervated (group II) compared to the non-denervated stomachs (group I). The treatment of the animals with MNNG induced the development of adenocarcinomas in non-denervated and denervated stomachs (groups III and IV, respectively) with a notable increase in the relative volume of the stroma, the frequency of reticular fibers and the inflammatory infiltrate that was more intense in group IV. An increase in the frequency of elastic fibers was observed in adenocarcinomas of denervated (group IV) compared to the non-denervated stomachs (group III) that showed degradation of these fibers. The development of lesions (groups III and IV) was also associated with an increase in the mast cell population, especially AB and AB-SAF positives, the latter mainly in the denervated group IV. Conclusions The results show a strong association in the morphological alteration of the ECM fibrillar components, the increased density of mast cells and the development of tumors induced by MNNG in the non-denervated rat stomach or denervated by BAC. This suggests that the study of extracellular and intracellular components of tumor microenvironment contributes to understanding of tumor biology by action of myenteric denervation.

Effects of enamel matrix derivative and transforming growth factor-β1 on human osteoblastic cells

Abstract Background Extracellular matrix proteins are key factors that influence the regenerative capacity of tissues. The objective of the present study was to evaluate the effects of enamel matrix derivative (EMD), TGF-β1, and the combination of both factors (EMD+TGF-β1) on human osteoblastic cell cultures. Methods Cells were obtained from alveolar bone of three adult patients using enzymatic digestion. Effects of EMD, TGF-β1, or a combination of both were analyzed on cell proliferation, bone sialoprotein (BSP), osteopontin (OPN) and alkaline phosphatase (ALP) immunodetection, total protein synthesis, ALP activity and bone-like nodule formation. Results All treatments significantly increased cell proliferation compared to the control group at 24 h and 4 days. At day 7, EMD group showed higher cell proliferation compared to TGF-β1, EMD + TGF-β1 and the control group. OPN was detected in the majority of the cells for all groups, whereas fluorescence intensities for ALP labeling were greater in the control than in treated groups; BSP was not detected in all groups. All treatments decreased ALP levels at 7 and 14 days and bone-like nodule formation at 21 days compared to the control group. Conclusions The exposure of human osteoblastic cells to EMD, TGF-β1 and the combination of factors in vitro supports the development of a less differentiated phenotype, with enhanced proliferative activity and total cell number, and reduced ALP activity levels and matrix mineralization.

Digestion of feed fractions and intake of heifers fed hydrolyzed sugarcane stored for different periods

The objective of this study was to evaluate, in Nellore heifers, intake and digestibility of hydrolyzed sugarcane stored for different periods. The experimental design used was a 4 × 4 Latin square, four diets, four Nellore heifers with ruminal cannulas (initial body weight 285.4±23.08 kg and average initial age 14 months) and four periods of 21 days. The diets were composed by fresh sugarcane (time zero) or hydrolyzed sugarcane with addition of 0.5% of hydrated lime, stored for 24, 48 or 72 hours, as the unique forage. Intake and digestibility of feed fractions, nitrogen balance, microbial synthesis efficiency, total number of ruminal protozoans and ammoniacal nitrogen did not significantly change by storing sugarcane with addition of 0.5% of hydrated lime. Sugarcane pH varied quadratically for storage time, with maximum pH of 7.02 after 24 hours from lime addition. Ruminal liquid pH values were higher for heifers fed fresh sugarcane, in comparison with those fed hydrolyzed sugarcane. Sugarcane treated with 0.5% of hydrated lime stored for up to 72 hours does not change ruminal digestion to alter the amount of feed consumed by pubescent Nellore heifers. Thus, lime is a viable technology, once it allows long-duration storage and bee control on treated forage, which contributes to animal feeding logistics.

Isolation of Microsporum gypseum in soil samples from different geographical regions of Brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase) and molecular sequencing of selected strains

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

Long-term type 1 diabetes impairs decidualization and extracellular matrix remodeling during early embryonic development in mice

Introduction: Endometrial decidualization and associated extracellular matrix (ECM) remodeling are critical events to the establishment of the maternal-fetal interface and successful pregnancy. Here, we investigated the impact of type 1 diabetes on these processes during early embryonic development, in order to contribute to the understanding of the maternal factors associated to diabetic embryopathies. Methods: Alloxan-induced diabetic Swiss female mice were bred after different periods of time to determine the effects of diabetes progression on the development of gestational complications. Furthermore, the analyses focused on decidual development as well as mRNA expression, protein deposition and ultrastructural organization of decidual ECM. Results: Decreased number of implantation sites and decidual dimensions were observed in the group mated 90-110 days after diabetes induction (D), but not in the 50-70D group. Picrosirius staining showed augmentation in the fibrillar collagen network in the 90e110D group and, following immunohistochemical examination, that this was associated with increase in types I and V collagens and decrease in type III collagen and collagen-associated proteoglycans biglycan and lumican. qPCR, however, demonstrated that only type I collagen mRNA levels were increased in the diabetic group. Alterations in the molecular ratio among distinct collagen types and proteoglycans were associated with abnormal collagen fibrillogenesis, analyzed by transmission electron microscopy. Conclusions: Our results support the concept that the development of pregnancy complications is directly related with duration of diabetes (progression of the disease), and that this is a consequence of both systemic factors (i.e. disturbed maternal endocrine-metabolic profile) and uterine factors, including impaired decidualization and ECM remodeling