Tissue distribution and depuration of the extracted hepatotoxic cyanotoxin microcystins in crucian carp (Carassius carassius) intraperitoneally injected at a sublethal dose

An acute toxicity experiment was conducted by intraperitoneal injection with a sublethal dose of extracted microcystins (MCs), 50 mu g MC-LR (where L = leucine and R = arginine) equivalent/kg body weight (BW), to examine tissue distribution and depuration of MCs in crucian carp (Carassius carassius). Liver to body weight ratio increased at 3, 12, 24, and 48 h postinjection compared with that at 0 h (p < 0.05). MC concentrations in various tissues and aquaria water were analyzed at 1, 3, 12, 24, 48, and 168 h postinjection using liquid chromatography coupled with mass spectrometry (LC-MS). The highest concentration of MCs (MC-RR + MC-LR) was found in blood, 2 -270 ng/g dry weight (DW), followed by heart (3 -100 ng/g DW) and kidney (13 -88 ng/g DW). MC levels were relatively low in liver, gonad, intestine, spleen, and brain. MC contents in gills, gallbladder, and muscle were below the limit of detection. Significant negative correlation was present between MC-RR concentration in blood and that in kidney, confirming that blood was important in the transportation of MC-RR to kidney for excretion. Rapid accumulation and slow degradation of MCs were observed in gonad, liver, intestine, spleen, and brain. Only 0.07% of injected MCs were detected in liver. The recovery of MCs in liver of crucian carp seemed to be dose dependent.

Co-electrospun poly(lactide-co-glycolide), gelatin, and elastin blends for tissue engineering scaffolds

In this study, we describe composite scaffolds composed of synthetic and natural materials with physicochemical properties suitable for tissue engineering applications. Fibrous scaffolds were co-electrospun from a blend of a synthetic biodegradable polymer (poly(lactic-co-glycolic acid), PLGA, 10% solution) and two natural proteins, gelatin (denatured collagen, 8% solution) and (x-elastin (20% solution) at ratios of 3:1:2 and 2:2:2 (v/v/v). The resulting PLGA-gelatin-elastin (PGE) fibers were homogeneous in appearance with an average diameter of 380 80 mn, which was considerably smaller than fibers made under identical conditions from the starting materials (PLGA, 780 +/- 200 nm; gelatin, 447 +/- 1.23 nm; elastin, 1060 170 nm). Upon hydration, PGE fibers swelled to an average fiber diameter of 963 +/- 132 nm, but did not disintegrate. Importantly, PGE scaffolds were stable in an aqueous environment without crosslinking, and were more elastic than those made of pure elastin fibers. To investigate the cytocompatibility of PGE, we cultured H9c2 rat cardiac myoblasts and rat bone marrow stromal cells (BMSCs) on fibrous PGE scaffolds. We found that myoblasts grew equally as well or slightly better on the scaffolds than on tissue-culture plastic. Microscopic evaluation confirmed that myoblasts reached confluence on the scaffold surfaces while simultaneously growing into the scaffolds.

Functional properties of cell-seeded three-dimensionally woven poly(epsilon-caprolactone) scaffolds for cartilage tissue engineering.

Articular cartilage possesses complex mechanical properties that provide healthy joints the ability to bear repeated loads and maintain smooth articulating surfaces over an entire lifetime. In this study, we utilized a fiber-reinforced composite scaffold designed to mimic the anisotropic, nonlinear, and viscoelastic biomechanical characteristics of native cartilage as the basis for developing functional tissue-engineered constructs. Three-dimensionally woven poly(epsilon-caprolactone) (PCL) scaffolds were encapsulated with a fibrin hydrogel, seeded with human adipose-derived stem cells, and cultured for 28 days in chondrogenic culture conditions. Biomechanical testing showed that PCL-based constructs exhibited baseline compressive and shear properties similar to those of native cartilage and maintained these properties throughout the culture period, while supporting the synthesis of a collagen-rich extracellular matrix. Further, constructs displayed an equilibrium coefficient of friction similar to that of native articular cartilage (mu(eq) approximately 0.1-0.3) over the prescribed culture period. Our findings show that three-dimensionally woven PCL-fibrin composite scaffolds can be produced with cartilage-like mechanical properties, and that these engineered properties can be maintained in culture while seeded stem cells regenerate a new, functional tissue construct.

Use of an insulating mask for controlling anisotropy in multilayer electrospun scaffolds for tissue engineering.

Tissue engineering of various musculoskeletal or cardiovascular tissues requires scaffolds with controllable mechanical anisotropy. However, native tissues also exhibit significant inhomogeneity in their mechanical properties, and the principal axes of anisotropy may vary with site or depth from the tissue surface. Thus, techniques to produce multilayered biomaterial scaffolds with controllable anisotropy may provide improved biomimetic properties for functional tissue replacements. In this study, poly(ε-caprolactone) scaffolds were electrospun onto a collecting electrode that was partially covered by rectangular or square shaped insulating masks. The use of a rectangular mask resulted in aligned scaffolds that were significantly stiffer in tension in the axial direction than the transverse direction at 0 strain (22.9 ± 1.3 MPa axial, 16.1 ± 0.9 MPa transverse), and at 0.1 strain (4.8 ± 0.3 MPa axial, 3.5 ± 0.2 MPa transverse). The unaligned scaffolds, produced using a square mask, did not show this anisotropy, with similar stiffness in the axial and transverse directions at 0 strain (19.7 ± 1.4 MPa axial, 20.8 ± 1.3 MPa transverse) and 0.1 strain (4.4 ± 0.2 MPa axial, 4.6 ± 0.3 MPa, transverse). Aligned scaffolds also induced alignment of adipose stem cells near the expected axis on aligned scaffolds (0.015 ± 0.056 rad), while on the unaligned scaffolds, their orientation showed more variation and was not along the expected axis (1.005 ± 0.225 rad). This method provides a novel means of creating multilayered electrospun scaffolds with controlled anisotropy for each layer, potentially providing a means to mimic the complex mechanical properties of various native tissues.

Diet-induced obesity alters the differentiation potential of stem cells isolated from bone marrow, adipose tissue and infrapatellar fat pad: the effects of free fatty acids.

INTRODUCTION: Obesity is a major risk factor for several musculoskeletal conditions that are characterized by an imbalance of tissue remodeling. Adult stem cells are closely associated with the remodeling and potential repair of several mesodermally derived tissues such as fat, bone and cartilage. We hypothesized that obesity would alter the frequency, proliferation, multipotency and immunophenotype of adult stem cells from a variety of tissues. MATERIALS AND METHODS: Bone marrow-derived mesenchymal stem cells (MSCs), subcutaneous adipose-derived stem cells (sqASCs) and infrapatellar fat pad-derived stem cells (IFP cells) were isolated from lean and high-fat diet-induced obese mice, and their cellular properties were examined. To test the hypothesis that changes in stem cell properties were due to the increased systemic levels of free fatty acids (FFAs), we further investigated the effects of FFAs on lean stem cells in vitro. RESULTS: Obese mice showed a trend toward increased prevalence of MSCs and sqASCs in the stromal tissues. While no significant differences in cell proliferation were observed in vitro, the differentiation potential of all types of stem cells was altered by obesity. MSCs from obese mice demonstrated decreased adipogenic, osteogenic and chondrogenic potential. Obese sqASCs and IFP cells showed increased adipogenic and osteogenic differentiation, but decreased chondrogenic ability. Obese MSCs also showed decreased CD105 and increased platelet-derived growth factor receptor α expression, consistent with decreased chondrogenic potential. FFA treatment of lean stem cells significantly altered their multipotency but did not completely recapitulate the properties of obese stem cells. CONCLUSIONS: These findings support the hypothesis that obesity alters the properties of adult stem cells in a manner that depends on the cell source. These effects may be regulated in part by increased levels of FFAs, but may involve other obesity-associated cytokines. These findings contribute to our understanding of mesenchymal tissue remodeling with obesity, as well as the development of autologous stem cell therapies for obese patients.

Peptide interfacial biomaterials improve endothelial cell adhesion and spreading on synthetic polyglycolic acid materials.

Resorbable scaffolds such as polyglycolic acid (PGA) are employed in a number of clinical and tissue engineering applications owing to their desirable property of allowing remodeling to form native tissue over time. However, native PGA does not promote endothelial cell adhesion. Here we describe a novel treatment with hetero-bifunctional peptide linkers, termed "interfacial biomaterials" (IFBMs), which are used to alter the surface of PGA to provide appropriate biological cues. IFBMs couple an affinity peptide for the material with a biologically active peptide that promotes desired cellular responses. One such PGA affinity peptide was coupled to the integrin binding domain, Arg-Gly-Asp (RGD), to build a chemically synthesized bimodular 27 amino acid peptide that mediated interactions between PGA and integrin receptors on endothelial cells. Quartz crystal microbalance with dissipation monitoring (QCMD) was used to determine the association constant (K (A) 1 x 10(7) M(-1)) and surface thickness (~3.5 nm). Cell binding studies indicated that IFBM efficiently mediated adhesion, spreading, and cytoskeletal organization of endothelial cells on PGA in an integrin-dependent manner. We show that the IFBM peptide promotes a 200% increase in endothelial cell binding to PGA as well as 70-120% increase in cell spreading from 30 to 60 minutes after plating.

Improved Equivalent Frame Analysis Method for Flat Plate Structures in Vicinity of Edge Columns

This paper proposes a modification to the ACI 318-02 equivalent frame method of analysis of reinforced concrete flat plate exterior panels. Two existing code methods were examined: ACI 318 and BS 8110. The derivation of the torsional stiffness of the edge strip as proposed by ACI 318 is examined and a more accurate estimate of this value is proposed, based on both theoretical analysis and experimental results. A series of 1/3-scale models of flat plate exterior panels have been tested. Unique experimental results were obtained by measuring strains in reinforcing bars at approximately 200 selected locations in the plate panel throughout the entire loading history. The measured strains were used to calculate curvature and, hence, bending moments; these were used along with moments in the columns to assess the accuracy of the equivalent frame methods. The proposed method leads to a more accurate prediction of the moments in the plate at the column front face, at the panel midspan, and in the edge column. Registered Subscribers: View the full article. This document is available as a free download to qualified members. An electronic (PDF) version is available for purchase and download. Click on the Order Now button to continue with the download.

Optimisation of radiotherapy for carcinoma of the parotid gland: a comparison of conventional, three-dimensional conformal, and intensity-modulated techniques

Background and purpose: To compare external beam radiotherapy techniques for parotid gland tumours using conventional radiotherapy (RT), three-dimensional conformal radiotherapy (3DCRT), and intensity-modulated radiotherapy (IMRT). To optimise the IMRT techniques, and to produce an IMRT class solution.Materials and methods: The planning target volume (PTV), contra-lateral parotid gland, oral cavity, brain-stem, brain and cochlea were outlined on CT planning scans of six patients with parotid gland tumours. Optimised conventional RT and 3DCRT plans were created and compared with inverse-planned IMRT dose distributions using dose-volume histograms. The aim was to reduce the radiation dose to organs at risk and improve the PTV dose distribution. A beam-direction optimisation algorithm was used to improve the dose distribution of the IMRT plans, and a class solution for parotid gland IMRT was investigated.Results: 3DCRT plans produced an equivalent PTV irradiation and reduced the dose to the cochlea, oral cavity, brain, and other normal tissues compared with conventional RT. IMRT further reduced the radiation dose to the cochlea and oral cavity compared with 3DCRT. For nine- and seven-field IMRT techniques, there was an increase in low-dose radiation to non-target tissue and the contra-lateral parotid gland. IMRT plans produced using three to five optimised intensity-modulated beam directions maintained the advantages of the more complex IMRT plans, and reduced the contra-lateral parotid gland dose to acceptable levels. Three- and four-field non-coplanar beam arrangements increased the volume of brain irradiated, and increased PTV dose inhomogeneity. A four-field class solution consisting of paired ipsilateral coplanar anterior and posterior oblique beams (15, 45, 145 and 170o from the anterior plane) was developed which maintained the benefits without the complexity of individual patient optimisation.Conclusions: For patients with parotid gland tumours, reduction in the radiation dose to critical normal tissues was demonstrated with 3DCRT compared with conventional RT. IMRT produced a further reduction in the dose to the cochlea and oral cavity. With nine and seven fields, the dose to the contra-lateral parotid gland was increased, but this was avoided by optimisation of the beam directions. The benefits of IMRT were maintained with three or four fields when the beam angles were optimised, but were also achieved using a four-field class solution. Clinical trials are required to confirm the clinical benefits of these improved dose distributions.

Autoradiographic and scintillation analysis of 5-aminolevulinic acid permeation through epithelialised tissue: Implications for topical photodynamic therapy of superficial gynaecological neoplasias

Purpose. Aminolevulinic acid (5-ALA) diffusion through both keratinised and non-keratinised tissue, used as a model tissue substrates, was evaluated, together with the depth of permeation and the concentration achieved following delivery from bioadhesive patch and proprietary cream formulations. Materials and Methods. Moisture-activated, bioadhesive patches loaded with 5-ALA at concentrations of 19.0, 38.0 and 50.0 mg cm(-2) and an o/w cream (20% w/w 5-ALA) were radiolabelled with C14 5-ALA and applied to excised human vaginal tissue and porcine skin. After 1, 2 and 4 h, tissue was sectioned in two orientations and the 5-ALA concentration at specific depths determined using autoradiography and liquid scintillation counting (LSC). Results. The stratum corneum was a significant barrier to 5-ALA permeation, with concentrations in tissue dependent on application time and drug loading. 5-ALA was detected at 6 mm using autoradiography after 2 h, with LSC showing phototoxic concentrations at 2.375 mm after 4 h of application. Inclusion of oleic acid and dimethyl sulphoxide in bioadhesive patches increased 5-ALA significantly in neonate porcine tissue, but only for patches cast from blends containing 5% w/w oleic acid. Conclusions. The bioadhesive patch described delivered 5-ALA to depths of at least 2.5 mm in tissue types indicative of vulval skin, suggesting that photodynamic therapy of deep vulval intraepithelial neoplasia is feasible using this means of bioadhesive 5-ALA delivery.

A new method of measuring plastic limit of fine materials

Index properties such as the liquid limit and plastic limit are widely used to evaluate certain geotechnical parameters of fine-grained soils. Measurement of the liquid limit is a mechanical process, and the possibility of errors occurring during measurement is not significant. However, this is not the case for plastic limit testing, despite the fact that the current method of measurement is embraced by many standards around the world. The method in question relies on a fairly crude procedure known widely as the ‘thread rolling' test, though it has been the subject of much criticism in recent years. It is essential that a new, more reliable method of measuring the plastic limit is developed using a mechanical process that is both consistent and easily reproducible. The work reported in this paper concerns the development of a new device to measure the plastic limit, based on the existing falling cone apparatus. The force required for the test is equivalent to the application of a 54 N fast-static load acting on the existing cone used in liquid limit measurements. The test is complete when the relevant water content of the soil specimen allows the cone to achieve a penetration of 20 mm. The new technique was used to measure the plastic limit of 16 different clays from around the world. The plastic limit measured using the new method identified reasonably well the water content at which the soil phase changes from the plastic to the semi-solid state. Further evaluation was undertaken by conducting plastic limit tests using the new method on selected samples and comparing the results with values reported by local site investigation laboratories. Again, reasonable agreement was found.

Elevated AGE-modified ApoB in sera of euglycemic, normolipidemic patients with atherosclerosis: relationship to tissue AGEs.:relationship to tissue AGEs

BACKGROUND: Advanced glycation endproducts (AGEs) are implicated in the pathogenesis of atherosclerotic vascular disease of diabetic and nondiabetic etiology. Recent research suggests that advanced glycation of ApoB contributes to the development of hyperlipidemia. AGE-specific receptors, expressed on vascular endothelium and mononuclear cells, may be involved in both the clearance of, and the inflammatory responses to AGEs. The aim of this study was to examine whether there is a relationship between serum AGE-ApoB and AGEs in arterial tissue of older normolipidemic nondiabetic patients with occlusive atherosclerotic disease, compared with age-matched and younger asymptomatic persons.

MATERIALS AND METHODS: Serum AGE-ApoB was measured by ELISA in 21 cardiac bypass patients. Furthermore, an AGE-specific monoclonal antibody, and polyclonal antibodies against anti-AGE-receptor (anti-AGE-R) 1 and 2 were used to explore the localization and distribution of AGEs and AGE-R immunoreactivity (IR) in arterial segments excised from these patients.

RESULTS: Serum AGE-ApoB levels were significantly elevated in the asymptomatic, older population, compared with those in young healthy persons (259 +/- 24 versus 180 +/- 21 AGE U/mg of ApoB, p < 0.01). Higher AGE-ApoB levels were observed in those patients with atherosclerosis (329 +/- 23 versus 259 +/- 24 AGE U/mg ApoB, p < 0.05). Comparisons of tissue AGE-collagen with serum AGE-ApoB levels showed a significant correlation (r = 0.707, p < 0.01). In early lesions, AGE-IR occurred mostly extracellularly. In fatty streaks and dense, cellular atheromatous lesions, AGE-IR was visible within lipid-containing smooth muscle cells and macrophages, while in late-stage, acellular plaques, AGE-IR occurred mostly extracellularly. AGE-R1 and -R2 were observed on vascular endothelial and smooth-muscle cells and on infiltrating mononuclear cells in the early-stage lesions, whereas in dense, late-stage plaques, they colocalized mostly with lipid-laden macrophages. On tissue sections, scoring of AGE-immunofluorescence correlated with tissue AGE and plasma AGE-ApoB.

CONCLUSIONS: (1) The correlation between arterial tissue AGEs and circulating AGE-ApoB suggests a causal link between AGE modification of lipoproteins and atherosclerosis. AGE-specific receptors may contribute to this process. (2) Serum AGE-ApoB may serve to predict atherosclerosis in asymptomatic patients.

The production of pig tissue sulphadimidine reference material

The production of stable homogeneous reference materials containing the antimicrobial agent sulphadimidine in pig tissue is described. These were commissioned by the Community Bureau of Reference (BCR), established by the Commission of the European Communities, to promote improvements in analytical accuracy and to ensure uniformity of results determined by member states. Sulphadimidine-containing tissue powders (400 vials each of muscle, liver and kidney) were prepared by orally dosing pigs with drug, producing lyophilized tissue powders and blending these with negative tissues from unmedicated animals to achieve target concentrations. Details of the production process, the stabilizing procedure developed and the analytical assessments of homogeneity and stability are given.