Note: Non-invasive optical method for rapid determination of alignment degree of oriented nanofibrous layers.

This paper presents a rapid non-destructive method that provides information on the anisotropic internal structure of nanofibrous layers. A laser beam of a wavelength of 632.8 nm is directed at and passes through a nanofibrous layer prepared by electrostatic spinning. Information about the structural arrangement of nanofibers in the layer is directly visible in the form of a diffraction image formed on a projection screen or obtained from measured intensities of the laser beam passing through the sample which are determined by the dependency of the angle of the main direction of polarization of the laser beam on the axis of alignment of nanofibers in the sample. Both optical methods were verified on Polyvinyl alcohol (PVA) nanofibrous layers (fiber diameter of 470 nm) with random, single-axis aligned and crossed structures. The obtained results match the results of commonly used methods which apply the analysis of electron microscope images. The presented simple method not only allows samples to be analysed much more rapidly and without damaging them but it also makes possible the analysis of much larger areas, up to several square millimetres, at the same time.

Multispecies biofilm in an artificial wound bed--A novel model for in vitro assessment of solid antimicrobial dressings.

Wound infections represent a major problem, particularly in patients with chronic wounds. Bacteria in the wound exist mainly in the form of biofilms and are thus resistant to most antibiotics and antimicrobials. A simple and cost-effective in vitro model of chronic wound biofilms applied for testing treatments and solid devices, especially wound dressings, is presented in this work. The method is based on the well-established Lubbock chronic wound biofilm transferred onto an artificial agar wound bed. The biofilm formed by four bacterial species (Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis and Pseudomonas aeruginosa) was stable for up to 48h post-transplant. The applicability of the model was evaluated by testing two common iodine wound treatments. These observations indicate that this method enables assessing the effects of treatments on established resilient wound biofilms and is clinically highly relevant.

Collection method for extra aligned nanofibers deposited by electrospinning.

Electrospinning is a very promising nanotechnology method, mostly thanks to industrial scale up ability, relatively low purchase cost, and huge variability. Nevertheless, its principal disadvantage is the way in which the individual nanofibers are randomly deposited while being collected onto a collector after passing through the chaotic whipping phase. Present and future applications of nanomaterials will need their precise, ordered intrinsic structures, or may even require accurately determined anisotropic properties because of significant improvement of material properties such as mechanical, electronic, optical, etc. This work demonstrates a novel and advanced collector including a sliding board mechanism. The resulting nanofibrous materials obtain extremely high orientation order parameter (S > 97%). Other important advantages of this technological method are discussed here in more detail.

Receptors and aging: structural selectivity of the rhamnose-receptor on fibroblasts as shown by Ca(2+)-mobilization and gene-expression profiles.

Qualitative and quantitative modifications of receptors were shown to play a key role in cell and tissue aging. We recently described the properties of a rhamnose-recognizing receptor on fibroblasts involved in the mediation of age-dependent functions of these cells. Using Ca(2+)-mobilization and DNA-microarrays we could show in the presence of rhamnose-rich oligo- and polysaccharides (RROPs) Ca(2+)-mobilization and changes in gene regulation. Here, we compared the effects of several RROPs, differing in their carbohydrate sequence and molecular weights, in normal human dermal fibroblasts (NHDFs). It appeared that different structural features were required for maximal effects on Ca(2+)-mobilization and gene-expression profiles. Maximal effect on Ca(2+) influx and intracellular free calcium regulation was exhibited by RROP-1, a 50 kDa average molecular weight polysaccharide, and RROP-3, a 5 kDa average molecular weight oligosaccharide with a different carbohydrate sequence. Maximal effect on gene-expression profiles was obtained with RROP-3. These results suggest the possibility of several different transmission pathways from the rhamnose-receptor to intracellular targets, differentially affecting these two intracellular functions, with potential consequences on aging. Although of only relative specificity, this receptor site exhibits a high affinity for rhamnose, absent from vertebrate glycoconjugates. The rhamnose-receptor might well represent an evolutionary conserved conformation of a prokaryote lectin.

Preparation and the kinetic stability of hyaluronan radiolabeled with 111In, 125I and 14C.

Three different procedures for the labeling of hyaluronan (HA) with (111)In, (125)I and (14)C radionuclides were compared, and the kinetic stability of radiolabeled HA under different conditions (saline, artificial gastric juice and plasma) was established. Modification of HA structure with bifunctional chelating agents (DTPA) or with the prosthetic group (tyramine or tyrosine) was essential prior (111)In and (125)I labeling. These chemical labeling techniques were fast, simple and inexpensive, and labeled agents with a high specific activity were obtained. The only disadvantage of these methods was the occurrence of unknown functional groups in the HA molecule requiring further characterization of the compound. Conversely, HA labeling with (14)C by biotechnological synthesis was found to be rather expensive and time-consuming process. Although, the final product (14)C-HA was identical to natural HA its low specific activity presents certain limitation for its application in biological experiments. Stability studies showed that (14)C-HA and (125)I-Tm-HA were stable in all studied mediums. In the case of (125)I-Trs-HA, stability slightly decreased in rat plasma and in artificial gastric juice with increasing time. The least stable was (111)In-DTPA-HA, which degraded completely after 48h in artificial gastric juice. Kinetic stability studies may provide primary information concerning the properties of radiolabeled HA in vitro, which is essential for the use and explanation of its behavior in biological experiments.

The effect of molecular weight on the biodistribution of hyaluronic acid radiolabeled with 111In after intravenous administration to rats.

Hyaluronic acid (HA), is a high molecular weight (HMW) glucosaminoglycan with significant acitivity, and which influences a number of physiological and pathological processes such as tumorogenesis, arthritis, etc. The aim of this study was to determine the difference in the biodistributional pathways of 111In-labeled diethylenetriaminepentaacetic acid-hyaluronic acid (111In-DTPA-HA) molecule of three different MWs (10, 100 and 450 kDA) in a rat model, and to determine possible relationships between the biodistribution and the MW of the investigated agent for future medical applications. 111In-DTPA-HA was prepared by mixing activated DTPA and activated HA, then adding 111InCl3 to the previously prepared mixture at pH 5,5 in an acetic buffer. Biodistributional studies were performed using 36 male Wistar rats aged 2 months and weighing 280 - 350 g. The radioactivity in the samples was measured via a radiometer and the radioactivity in the different organs, blood, plasma and urine was determined. It was found that 50-54% for 10 and 100 kDa and 80% for 450 kDa of the administered dose of radiolabel was present in the liver after 5 min. Other organs show no significant increase during the experimental period. The elimination of the radiolabel was mostly renal and in low molecular weight (LMW) form. Radioactivity remained in liver throughout the 72h experimental period. A difference in the biodistribution of 450 kDa and LMW radiolabeled molecules was found. Higher amounts of radiolabel were taken up by the liver when the 450 kDa molecule was used. LMW fractions were found in the urine, and could have been a product of non-enzymatic cleavage. The extended retention of radiolabel in the liver could be related to changes in the polarity of DTPA-HA molecules.

The alpha-L-Rhamnose recognizing lectin site of human dermal fibroblasts functions as a signal transducer: modulation of Ca2+ fluxes and gene expression.

An alpha-l-Rhamnose specific lectin site was described on human skin keratinocytes and fibrobasts. The addition of Rhamnose-rich oligo- and polysaccharides (RROPs) to fibroblasts has been shown to stimulate cell proliferation and increase extracellular matrix biosynthesis, suggesting that this lectin site functions as a "true" receptor transmitting messages to the cell interior. It was confirmed here that addition of the Rhamnose-rich polysaccharide, RROP-1, to normal human dermal fibroblasts (NHDFs) and human endothelial cells produced a dose-dependent stimulation of the calcium-signaling pathway, inducing fast and transient increases in Ca2+ influx and intracellular free Ca2+ level. The Rhamnose-rich oligosaccharide RROP-3 as well as l-Rhamnose alone were also able to trigger similar intracellular free Ca2+ concentration increases in NHDFs. Moreover, the recording of the RROP-1-induced modification of the gene-expression profile in fibroblasts showed that this polysaccharide triggered a down-regulation of the expression of several growth factors, adhesion molecules and extracellular matrix proteins involved in pro-tumoral activity and/or fibrotic processes. These results further support the hypothesis of a receptor function for the Rhamnose-recognizing lectin site in fibroblasts. Anti-fibrotic and anti-tumoral potential of RROP-1 remains to be further explored.

Effect of advanced glycation endproducts on gene expression profiles of human dermal fibroblasts.

The Maillard reaction and its end products, AGE-s (Advanced Glycation End products) are rightly considered as one of the important mechanisms of post-translational tissue modifications with aging. We studied the effect of two AGE-products prepared by the glycation of lysozyme and of BSA, on the expression profile of a large number of genes potentially involved in the above mentioned effects of AGE-s. The two AGE-products were added to human skin fibroblasts and gene expression profiles investigated using microarrays. Among the large number of genes monitored the expression of 16 genes was modified by each AGE-preparations, half of them only by both of them. Out of these 16 genes, 12 were more strongly affected, again not all the same for both preparations. Both of them upregulated MMP and serpin-expression and downregulated some of the collagen-chain coding genes, as well as the cadherin- and fibronectin genes. The BSA-AGE preparation downregulated 10 of the 12 genes strongly affected, only the serpin-1 and MMP-9 genes were upregulated. The lysozyme-AGE preparation upregulated selectively the genes coding for acid phosphatase (ACP), integrin chain alpha5 (ITGA5) and thrombospondin (THBS) which were unaffected by the BSA-AGE preparation. It was shown previously that the lysozyme-AGE strongly increased the rate of proliferation and also cell death, much more than the BSA-AGE preparation. These differences between these two AGE-preparations tested suggest the possibility of different receptor-mediated transmission pathways activated by these two preparations. Most of the gene-expression modifications are in agreement with biological effects of Maillard products, especially interference with normal tissue structure and increased tissue destruction.

Pharmacological properties of rhamnose-rich polysaccharides, potential interest in age-dependent alterations of connectives tissues.

Rhamnose-rich oligo- and polysaccharides (RROPs) were tested for their potential pharmacological properties using human skin fibroblasts in serial cultures. The substances tested were shown to stimulate cell proliferation, decrease elastase-type activity, stimulate collagen biosynthesis, and protect hyaluronan against free radical mediated degradation. These reactions appear to be triggered by the mediation of a specific alpha-L-rhamnose recognizing lectin-site acting as a receptor, transmitting signals to the cell-interior. The rapid increase of intracellular free calcium after addition of RROP-1 and preliminary data using micro arrays appear also to confirm this contention.

[Sodium hyaluronate and an iodine complex--Hyiodine--new method of diabetic defects treatment]. / Komplex hyaluronanu a jódu--hyiodine--nova metoda pri terapii diabetických defektu.

UNLABELLED: Complicated diabetic defects are difficult to heal and frequently result in leg amputation. We have developed a new and unique system for wound treatment, which is based on combination of high molecular weight sodium hyaluronate with an iodine complex--Hyiodine. The aim of our study was to assess the effect of this new method of wound dressing on infected diabetic defects healing. METHODS: The effect of Hyiodine was studied on 22 patients suffering from complicated foot diabetic wounds. Hyiodine was either spread directly over the wound, or (more frequently) gauze was immersed in Hyiodine and then put on/into the wound. Then several layers of dry gauze covered the wound. RESULTS: Within 2-6 weeks after the onset of treatment all but two defects were filled with granulation tissue. Complete healing was evident in 18 patients within 6-20 weeks after the start of treatment, depending on the wound character, localization and extent. Two patients are still treated by Hyiodine, and significant improvement is apparent on their wound. Treatment was not successful in two subjects with ischemic defects due to simultaneous arterial occlusion. CONCLUSIONS: We can conclude that the hyaluronan-iodine complex Hyiodine is efficient method for treatment of difficult to heal diabetic defects without complete arterial occlusion.

The effects of hyaluronan on bone resorption and bone mineral density in a rat model of estrogen deficiency-induced osteopenia.

Hyaluronan, or hyaluronic acid (HA), is an essential component of extracellular matrices. HA of appropriate molecular weight and concentration can induce osteoblast differentiation and bone formation in vitro. The aim of our study was to evaluate the effects of HA of different molecular weights on ovariectomy (OVX)-induced bone loss in rats. Adult female Sprague Dawley rats were subjected to bilateral OVX or sham surgical procedure (sham). OVX rats were treated with: HA of molecular weight of 0.75 MDa at a dose of 1 mg/kg/day and with HA of molecular weight of 1.62 MDa at a dose of both 0.5 mg/kg/day and 1 mg/kg/day. HA was applied orally once a day during the 8-week period after ovariectomy. Body weight, urinary pyridinoline (Pyr), deoxypyridinoline (DPyr) corrected for urinary creatinine, serum nitrite/nitrate concentrations and whole body and femoral bone mineral density (BMD) were measured. HA treatment had no effect on the body weight gain in OVX rats. Excretion of urinary Pyr and Dpyr significantly increased in OVX rats compared to sham controls. The higher molecular weight HA (1.62 MDa) significantly reduced urinary Pyr and DPyr concentrations measured on day 28 after ovariectomy (p < 0.001). Serum concentrations of nitric oxide metabolites, nitrite/nitrate significantly decreased in OVX rats in comparison with sham controls (p < 0.001). HA of both 0.75 MDa and 1.62 MDa molecular weights significantly enhanced serum nitrite/nitrate concentrations in OVX rats. There was a clear reduction of whole body and femoral BMD in untreated OVX rats. The higher molecular weight HA decreased both whole body and femoral BMD loss. Our results show that orally applied HA of high molecular weight (1.62 MDa) inhibits bone resorption and provides a protective effect on bone density in ovariectomized rats.

Elastin content in human fibrotic and cirrhotic liver.

Human liver tissue obtained at autopsy was extracted with hot 0.1 M NaOH. Determination of amino acids desmosine and isodesmosine was used to quantify elastin in the insoluble residue. Hydroxyproline content was used as an index of collagen content in the liver. Hydroxyproline content was twofold in fibrotic liver and threefold in cirrhotic liver when compared to normal liver, while the sum of desmosine and isodesmosine increased threefold and sixfold, respectively. Elastin content is low in normal liver when compared to collagen but appears to grow much faster in diseased liver.

Elastin powder produced for cosmetic purposes does not affect formation of granulation tissue.

A suspension of elastin powder that is produced for cosmetic purposes has been applied on open skin wounds made in rats. Elastin particles (diameter smaller than 0.1 mm) prepared by extraction of bovine nuchal ligament with alkali had no effect on wet weight and dry weight of granulation tissue formed in the wound within 6 days. Hydroxyproline content which indicates collagen content of the granulation tissue was not also changed.

On the influence of the substrate (elastin) in elastase-alpha 1 antitrypsin interactions.

Evidence is presented showing that elastin(s) influences the interactions between porcine pancreatic elastase and its main plasma inhibitor alpha 1-proteinase inhibitor (e.g. alpha 1 antitrypsin). The rate constant of association (kon) between the proteinase and alpha 1 antitrypsin is decreased from 2.8 10(5) M-1 sec-1 to 1.24 10(5) M-1 sec-1 in presence of 1 mg/ml of insoluble elastin and 0.83 10(5) M sec-1 in presence of the same concentration of alpha-elastin. With both elastins this kon decrease was found concentration dependent indicating that these compounds compete in a similar fashion with alpha 1 AT for the binding of elastase. Such an effect was not observed when elastin(s) was replaced by bovine serum albumin. Experiments were designed as previously published (B. Robert et al. 1974) in order to evaluate the capacity of alpha 1 AT to inhibit preadsorbed elastase on to insoluble elastin and also diffusible protease molecules. It was found that even at concentrations far exceeding the equivalence with elastase, alpha 1 AT was only partially able to inhibit both preadsorbed enzyme (approximately 50 per cent) and diffusible protease (approximately 90 per cent).