A study of the damage behaviour of porcine intervertebral discs in a bioreactor environment.

The intervertebral discs are cartilaginous, articulating structures that lie between vertebral bodies, allowing flexibility, transmission, modification, and also distribution of the forces to the spinal column. Disc degeneration is characterised by progressive loss of disc height and exaggerated radial bulging. Therefore, the spine becomes shorter, stiffer, and less mobile. In the last several decades, there is a strong need for a tissue engineering strategy that alleviates pain and restores spine function by directly addressing the underlying biological causes of disc degeneration. Numerous studies that are currently showing potential have been conducted on developing regenerative and reparative strategies for treating this condition. In this study, to numerically describe the anisotropic mechanical damage behaviour of discs, the pseudo-elastic damage model was applied. To experimentally picture the biomechanical response of discs and to study the damage mechanisms as well as the spinal disc herniation, a special bioreactor was evolved. The specimens were obtained from pigs aged six months. A total of eight functional spine units were taken from porcine lumbar spines (L1-L2). Firstly, the experiments were performed by using long-term cyclic uniaxial compression tests. Secondly, the mean value of experimental results with consideration of the different shapes and sizes of the samples was calculated. Afterwards, the experimental results were compared with outcomes of numerical simulations.

A comparative study of mechanical properties of fresh and frozen-thawed porcine intervertebral discs in a bioreactor environment.

Limited availability of fresh intervertebral discs (IVDs) necessitates the use of frozen-thawed samples in biomechanical investigations. Several authors have reported the mechanical properties of frozen-thawed IVDs, but few studies have investigated the effects of storage temperature and cooling rate. The validity and reliability of the applied freezing-thawing procedures are still relatively unknown. The study aims to compare the mechanical responses of fresh porcine IVDs and porcine IVDs, which were frozen at different freezing temperatures with a controlled-rate cooling process and then thawed. The specimens were obtained from pigs aged 6 months. A total of 18 functional spine units (FSUs) were taken from seven porcine lumbar spines (L1-L6). The specimens were then split into three groups. The first group was tested fresh immediately and the other two groups were frozen at the same cooling rate and stored at -20°C and -80°C, respectively, before testing. The period of storage ranged between 12 and 43 days. The frozen specimens were thawed for 9h at room temperature before the tests. A special IVD bioreactor, which maintained the realistic behaviour of IVDs under various loading conditions, was developed. The analysis of variance (ANOVA) was used to determine if the observed results were statistically significant. The results indicated that frozen storage at -20°C decreases the comprehensive stiffness. In contrast, freezing to -80°C did not seem to have any effect on the mechanical properties of IVDs. No significant differences in outcome were observed for the samples, which had different spine levels. The study confirmed the reliability and usability of frozen-thawed samples stored at -80°C for biomechanical investigations.

Effects of osteoarthritis and pathological walking on contact stresses in femoral cartilage.

Osteoarthritis is a widespread abnormality in synovial joints leading to increasing pain and potential work disability in middle-aged and older populations. A primary cause of osteoarthritis is related to damages from high local stresses combined with insufficient self-healing of cartilage. In this framework, it is the goal of the present contribution to offer a thermodynamically consistent simulation of a highly anisotropic, heterogeneous, osmotic swelling and poroviscoelastic model of healthy and osteoarthritic articular cartilage based on the Theory of Porous Media. Physiological and pathological loading patterns are included by means of multi-body system calculations on patients. The contact stresses at the cartilage surface are represented by means of three-dimensional and simplified stereographic views of the femoral head. For normal walking, the stress peaks are higher in the degenerated case than in the healthy case. Interestingly, pathological walking combined with degenerated cartilage tissue minimises the occurrence of high local stresses.

Arsenic and other trace elements in two catfish species from Paranaguá Estuarine Complex, Paraná, Brazil.

Concentrations of arsenic and four additional trace elements (Cu, Cr, Ni, and Zn) were determined by inductively coupled plasma-optical emission spectrometry in the muscular tissue of the yellow catfish (Cathorops spixii) and the urutu catfish (Genidens genidens) from Paranaguá Estuarine Complex, Brazil (PEC). The PEC can be characterized by an environment of high ecological and economic importance in which preserved areas of rainforest and mangroves coexist with urban activities as ports and industries. The average concentrations (in milligram per kilogram dry weight) of elements in the muscle tissue of C. spixii are as follows: Zn (31), As (17), Cu (1.17), Cr (0.62), and Ni (0.28). Similar concentrations could be found in G. genidens with exception of As: Zn (36), As (4.78), Cu (1.14), Cr (0.51), and Ni (0.14). Fish from the geographic northern rural region (Guaraqueçaba-Benito) display higher As concentrations in the muscle tissues than fish found in the south-western (urban) part of the PEC. An international comparison of muscle tissue concentrations of trace elements in fish was made. Except for Ni in C. spixii, a tendency of decrease in element concentration with increasing size (age) of the fish could be observed. According to the National Health Surveillance Agency of Brazil, levels of Cr and As exceeded the permissible limits for seafood. An estimation of the provisional tolerable weekly intake of As was calculated with 109 % for C. spixii and with 29 % for G. genidens.

First study on anthropogenic Pt, Pd, and Rh levels in soils from major avenues of São Paulo City, Brazil.

Over the last years, investigations on the increase of platinum (Pt), palladium (Pd), and rhodium (Rh) levels in urban environments of big cities all over the world - especially to catalytic converters emissions - have been grown up enormously. São Paulo City is the 6th largest megacity in the world having about 20 million inhabitants and an ever increasing seven million motor vehicle fleet. In spite of this, there has never been an investigation regarding Pt, Pd, and Rh levels in the city. In the present study, Pt, Pd, and Rh concentrations were determined in soils adjacent to seven main high-density traffic avenues in the metropolitan region of São Paulo City. Inductively coupled plasma mass spectrometry was employed - after ultrasound-assisted aqua regia leaching - as analytical technigue. The results showed concentration levels up to 378 ng g(-1) for Pd, 208 ng g(-1) for Pt, and 0.2 to 45 ng g(-1) for Rh. These levels are much higher than those considered for the geochemical background of soils, indicating a catalytic converter source. Due to the different Pt/Pd/Rh ratio in Brazilian automobile catalytic converters, lower levels of Pt/Pd ratios compared with other similar studies were observed. The obtained results are the first data for monitoring Pt, Pd, and Rh pollution in São Paulo City soils.

An extended biphasic model for charged hydrated tissues with application to the intervertebral disc.

Finite element models for hydrated soft biological tissue are numerous but often exhibit certain essential deficiencies concerning the reproduction of relevant mechanical and electro-chemical responses. As a matter of fact, singlephasic models can never predict the interstitial fluid flow or related effects like osmosis. Quite a few models have more than one constituent, but are often restricted to the small-strain domain, are not capable of capturing the intrinsic viscoelasticity of the solid skeleton, or do not account for a collagen fibre reinforcement. It is the goal of this contribution to overcome these drawbacks and to present a thermodynamically consistent model, which is formulated in a very general way in order to reproduce the behaviour of almost any charged hydrated tissue. Herein, the Theory of Porous Media (TPM) is applied in combination with polyconvex Ogden-type material laws describing the anisotropic and intrinsically viscoelastic behaviour of the solid matrix on the basis of a generalised Maxwell model. Moreover, other features like the deformation-dependent permeability, the possibility to include inhomogeneities like varying fibre alignment and behaviour, or osmotic effects based on the simplifying assumption of Lanir are also included. Finally, the human intervertebral disc is chosen as a representative for complex soft biological tissue behaviour. In this regard, two numerical examples will be presented with focus on the viscoelastic and osmotic capacity of the model.

Heavy metals in emergent trees and pioneers from tropical forest with special reference to forest fires and local pollution sources in Sarawak, Malaysia.

Leaf samples of tropical trees, i.e. Dryobalanops lanceolata (Kapur paji), Dipterocarpaceae and Macaranga spp. (Mahang), Euphorbiaceae were analyzed for 21 chemical elements. The pioneer Macaranga spp. exhibited higher concentrations for the majority of elements compared to the emergent species of Dryobalanops lanceolata, which was attributed to the higher physiological activity of the fast growing pioneer species compared to emergent trees. Lead showed rather high concentrations in several samples from the Bakam re-forestation site. This is suggested to be caused by emissions through brick manufacturing and related activities in the vicinity. A comparison of Dryobalanops lanceolata samples collected in 1993, 1995 and 1997 in the Lambir Hills National Park revealed that certain heavy metals, i.e. Co, Cu, Mn, Ni, Pb and Ti showed higher values in 1997 compared to the previous years, which could indicate an atmospheric input from the haze caused by the extensive forest fires raging in Borneo and other parts of Southeast Asia.

A linear viscoelastic biphasic model for soft tissues based on the Theory of Porous Media.

Based on the Theory of Porous Media (mixture theories extended by the concept of volume fractions), a model describing the mechanical behavior of hydrated soft tissues such as articular cartilage is presented. As usual, the tissue will be modeled as a materially incompressible binary medium of one linear viscoelastic porous solid skeleton saturated by a single viscous pore-fluid. The contribution of this paper is to combine a descriptive representation of the linear viscoelasticity law for the organic solid matrix with an efficient numerical treatment of the strongly coupled solid-fluid problem. Furthermore, deformation-dependent permeability effects are considered. Within the finite element method (FEM), the weak forms of the governing model equations are set up in a system of differential algebraic equations (DAE) in time. Thus, appropriate embedded error-controlled time integration methods can be applied that allow for a reliable and efficient numerical treatment of complex initial boundary-value problems. The applicability and the efficiency of the presented model are demonstrated within canonical, numerical examples, which reveal the influence of the intrinsic dissipation on the general behavior of hydrated soft tissues, exemplarily on articular cartilage.

On the microwave digestion of soils and sediments for determination of lanthanides and some toxic and essential elements by inductively coupled plasma source mass spectrometry.

Four digestion procedures for microwave dissolution of soils and sediments are investigated. Accurate results (accuracy better than 10%) for lanthanides, Be, Bi, Te, Th, U and Y are obtained only after an overnight stay with a mixture of 2 ml 48% HF and 2 ml 65% HNO(3) and afterwards stepwise microwave digestion with 1 ml 48% HF and 10 ml 5% H(3)BO(3). Only As is not be to be determined by this procedure due to significant interferences from Eu, Nd and Sm. For all other elements, accurate results are obtained by all four procedures.

Distribution of lanthanoids, Be, Bi, Ga, Te, Tl, Th and U on the territory of Bulgaria using Populus nigra 'Italica' as an indicator.

The concentrations of lanthanoids, Be, Bi,Ga, Te, Tl, Th and U have been determined using ICP-MS for 100 standardized samples of poplar leaves collected from the territory of Bulgaria. The investigated elements are log-normally distributed on the territory. Using cluster analysis of the analytical data the samples were grouped according soil type on which the plants are growing.

Effects of endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part III: Cyproterone acetate and vinclozolin as antiandrogens.

The effects of suspected endocrine disrupting chemicals on freshwater and marine prosobranch species were analysed in laboratory experiments. In this last of three publications, the responses of the fresh water snail Marisa cornuarietis and of two marine prosobranchs (Nucella lapillus, Nassarius (Hinia) reticulatus) to the antiandrogenic model compounds cyproterone acetate (CPA) and vinclozolin (VZ) are presented. The snails were exposed to nominal CPA concentrations of 1.25 mg/L alone and simultaneously to a potent synthetic estrogen (ethinylestradiol), androgen (methyltestosterone) or an indirectly acting xeno-androgen (tributyltin) in experiments with adult specimens and in a life cycle test for 12 months. Marisa and Nucella were furthermore exposed to nominal concentrations of 0.03-1.0 microgram VZ/L for up to 5 months. The antiandrogens induced a number of biological responses in all three species. The length of the penis and of accessory male sex organs (e.g., penis sheath, prostate) were significantly reduced. For Marisa, this effect occurred only in sexually immature specimens and was reversible as the males attained puberty. Typical androgen-mediated responses (imposex development, delayed spermatogenesis, tubulus necrosis of the testis with orchitis and Leydig cell hyperplasia) were partially or totally suppressed by a simultaneous administration of CPA. In the two marine species even adult, sexually mature males responded to antiandrogens with a reduction of the male sex organs and an advancement of the sexual repose phase. The results for CPA and VZ are compared with the effects of an exposure to xeno-estrogens (bisphenol A, octylphenol) and xeno-androgens (triphenyltin, tributyltin) in the same species. Each group of endocrine disruptors induces a characteristic set of toxicological effects in prosobranch snails which can be used as endpoints in an organismic invertebrate test for the identification of endocrine mimetic test compounds. Estrogens cause primarily an induction of superfemales resulting in an increased female mortality by the enhancement of spawning mass and egg production. The main effects of androgens are a virilization of females by imposex development and a marked decrease of the fecundity. Compared with estrogens and androgens, the antiandrogen responses seem to be less drastic and might have--in contrast to the two other disruptor classes--no biologically significant effects at the population level.

The Biological System of the Elements (BSE). Part II: a theoretical model for establishing the essentiality of chemical elements. The application of stoichiometric network analysis to the biological system of the elements

Stoichiometric Network Analysis (SNA), originally developed by the Canadian chemist Bruce L. Clarke during the 1970s, provides a most efficient means of reducing the background topology of complex interaction networks to some skeleton topology around which systems dynamics can be understood without jeopardising insight into complex dynamics by over- or miss-simplification. Since it focuses on the corresponding autocatalytic (AC) features of a feedback system as those which control overall behaviour to some extent, SNA deals with reaction kinetics in and beyond chemistry, e.g. with nuclear reactions. It is therefore quite straightforward to apply this manner of simplification, which in turn is supported by a number of mathematical theorems on systems behaviour and properties of AC cycles, to biological systems although their 'full' complexity may not even be assessed in the yet rare cases of complete genetic sequencing. Assuming there is a relationship between the kinds of metal or metalloid species and key biological/biochemical transformations to be promoted with their aid--this relationship being the subject of bio-inorganic chemistry--and that biochemistry is, in effect, about systems which can reproduce and thus behave autocatalytically, one can expect SNA to yield formally sound statements on basic features of biology and biochemistry too. If we sum up the facts and considerations concerning essentiality or possible essentiality in a biological system of elements (Markert, 1994), this means joining the triangular representation of BSE, including statements on (the degree of biological) evolution and aggregation levels, to SNA treatment of autocatalysis within hierarchical systems from metalloenzymes to entire biocoenoses. Arguments using preferred cluster sizes and aggregation tendencies from coordination chemistry are then employed to circumscribe possible functions within the BSE. They are also extended to metals hitherto not known to be essential, such as tellurium or scandium.

Deposition and disease: a moss monitoring project as an approach to ascertaining potential connections.

In the years 1995 and 1996 the atmospheric deposition of elements in the EuroRegion Neisse (ERN) was determined in a biomonitoring project using mosses. The mosses Pleurozium schreberi and Polytrichum formosum were chosen as biomonitors because of their wide distribution in the area studied. The moss samples were analysed by ICP-MS and ICP-OES for their concentrations of 37 chemical elements. The results were shown in the form of maps. The data from the moss monitoring project served as a basis for determining those elements in the deposited material that promote the occurrence of disease. This was done by correlating the figures for the various diseases with the appropriate element concentrations in the mosses. Indications were found that a connection exists between the thallium content of mosses and the occurrence of cardiovascular disease and between Ce, Fe, Ga and Ge levels in the mosses and the incidence of diseases of the respiratory system.

Effects of endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part II: Triphenyltin as a xeno-androgen.

In laboratory experiments the effects of suspected endocrine disrupting chemicals on freshwater and marine prosobranch species were analysed. In this second of three publications the responses of the freshwater ramshorn snail Marisa cornuarietis and of two marine prosobranchs (the dogwhelk Nucella lapillus and the netted whelk Hinia reticulata) to the xeno-androgenic model compound triphenyltin (TPT) are presented. Marisa and Nucella were exposed via water (nominal concentrations 5-500 ng TPT-Sn/L) and Hinia via sediments (nominal concentrations 50-500 micrograms TPT-Sn/kg dry wt.) for up to 4 months. Female ramshorn snails but not the two marine species developed imposex in a time and concentration dependent manner (EC10 4 months: 12.3 ng TPT-Sn/L) with a comparable intensity as described for tributyltin. TPT reduced furthermore the fecundity of Marisa at lower concentrations (EC10 4 months: 5.59 ng TPT-Sn/L) with a complete inhibition of spawning at nominal concentrations > or = 250 ng TPT-Sn/L (mean measured +/- SD: > or = 163 +/- 97.0 ng TPT-Sn/L). The extension of the pallial sex organs (penis with accessory structures and prostate gland) of male ramshorn snails and dogwhelks were reduced by up to 25% compared to the control but not in netted whelks. Histopathological analyses for M. cornuarietis and H. reticulata provide evidence for a marked impairment of spermatogenesis (both species) and oogenesis (only netted whelks). The test compound induced a highly significant and concentration independent increase in the incidence of hyperplasia on gills, osphradia and other organs in the mantle cavity of N. lapillus indicating a carcinogenic potential of TPT. The results show that prosobranchs are sensitive to endocrine disruption at environmentally relevant concentrations of TPT. Also, M. cornuarietis is a promising candidate for a future organismic invertebrate system to identify endocrine-mimetic test compounds.

Effects of endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part I: Bisphenol A and octylphenol as xeno-estrogens.

The effects of suspected endocrine disrupting chemicals on freshwater and marine prosobranch species were analysed in laboratory experiments. In this first publication, the responses of the freshwater snail Marisa cornuarietis and of the marine prosobranch Nucella lapillus to the xeno-estrogenic model compounds bisphenol A (BPA) and octylphenol (OP) are presented at nominal concentration ranges between 1 and 100 micrograms/L. Marisa was exposed during 5 months using adult specimens and in a complete life-cycle test for 12 months. In both experiments, the xeno-estrogens induced a complex syndrome of alterations in female Marisa referred to as "superfemales" at the lowest concentrations. Affected specimens were characterised by the formation of additional female organs, an enlargement of the accessory pallial sex glands, gross malformations of the pallial oviduct section resulting in an increased female mortality, and a massive stimulation of oocyte and spawning mass production. The effects of BPA and OP were comparable at the same nominal concentrations. An exposure to OP resulted in inverted U-type concentration response relationships for egg and spawning mass production. Adult Nucella from the field were tested for three months in the laboratory. As in Marisa, superfemales with enlarged accessory pallial sex glands and an enhancement of oocyte production were observed. No oviduct malformations were found probably due to species differences in the gross anatomical structure of the pallial oviduct. A lower percentage of exposed specimens had ripe sperm stored in their vesicula seminalis and additionally male Nucella exhibited a reduced length of penis and prostate gland when compared to the control. Because statistically significant effects were observed at the lowest nominal test concentrations (1 microgram BPA or OP/L), it can be assumed that even lower concentrations may have a negative impact on the snails. The results show that prosobranchs are sensitive to endocrine disruption at environmentally relevant concentrations and that especially M. cornuarietis is a promising candidate for a future organismic invertebrate model to identify endocrine-mimetic test compounds.

Analysis of phytochelatins in plant matrices by pre-column derivatization, high-performance liquid chromatography and fluorescence-detection.

A sensitive method for the determination of phytochelatins in plant matrices by pre-column derivatization with monobromobimane (mBrB) and high-performance liquid chromatography (HPLC) on reversed phases and fluorescence-detection has been developed and applied to cucumber sprouts (Cucumis sativus) treated with cadmium and to the water moss Fontinalis antipyretica (Cd in environmentally-relevant concentrations). Whereas phytochelatins were found in the Cd-treated sprouts, no phytochelatins were detected in Fontinalis anitipyretica.

Investigations on the water content of candidate reference material CRM 679 cabbage powder.

The determination of the residual water content by thermal methods and especially by Karl Fischer titration in a candidate reference material of the European Commission (candidate CRM 679 cabbage powder) is described and discussed.

The German heavy metal survey by means of mosses.

This is the first attempt to determine pollution with metals throughout the Federal Republic of Germany by analysing moss samples. Samples of Pleurozium schreberi, Scleropodium purum, Hypnum cupessiforme and Hylocomium splendens were collected at 593 sites and analysed by ICP-AES and AAS for the elements As, Cd, Cr, Cu, Fe, Ni, Pb, V and Zn. Citrus leaves and pine needles were used as reference materials to ensure the quality of the results. In many cases it was possible to trace the areas affected by known sources of heavy-metal emissions in addition to isolated local increases in the values. The moss monitoring programme showed up the highly industrialized and urban locations such as the Ruhr, parts of the Saarland and Baden-Wurttemberg and large areas of eastern Germany. Lower levels of many elements were found in wide stretches of Lower Saxony and Bavaria. The results largely reflect the pollution patterns found in these areas. On the other hand, expected correlations between the effects of traffic (e.g. Pb) and concentrations in moss could not be demonstrated with certainty. The element data yielded by this project are Germany's contribution to the European project 'Atmospheric Heavy Metal Deposition in Europe -- Estimations based on Moss Analysis'.

Tributyltin biomonitoring using prosobranchs as sentinel organisms.

Tributyltin (TBT) compounds, some of the most toxic xenobiotics, produce a variety of pathological reactions in animals. A reliable biomonitoring method to assess the degree of environmental TBT pollution has been described based on investigations of virilization phenomena in prosobranch snails (Mollusca: Gastropoda). Examples are the imposex phenomenon in marine and freshwater species, the intersex reaction in littorinids and the reduction of female sexual glands and offspring numbers in further species resulting mainly in a sterilization of females. The degree of imposex or intersex in populations is determined by different biomonitoring indices which allow to assess the TBT pollution of the environment at low costs with high precision. The effectiveness of TBT legislations is analysed by extensive surveys in France and Ireland indicating that there is still a continuing threat to sensitive marine organisms. TBT disturbs the biosynthesis of steroid hormones on the level of estrogen biosynthesis. The observed virilization phenomena seem due to an inhibition of the cytochrome P-450 dependent aromatase by this organotin compound.

Moss interspecies comparisons in trace element concentrations.

Within the framework of a European-scaled moss survey, various moss species were sampled throughout The Netherlands [NL], Germany [D], and Switserland [CH], and used in moss interspecies comparisons of elemental concentrations. Moss species considered were Pleurozium schreberi [NL,D,CH], Brachythecium rutabulum [NL], Hypnum cupressiforme [D,CH], Hylocomium splendens [D,CH], and Scleropodium purum [D]. Element analysis was carried out directly (The Netherlands, Instrumental Neutron Activation Analysis: As, Br, Ce, Cr, Cs, Fe, La, Mn, Na, Rb, Sc, Se, Sm, Th, Ti, V and Zn), or after sample digestion (The Netherlands, ICP-MS: Pb, Germany, AAS/ICP-AES: Cd, Cr, Cu, Fe, Ni, Pb, Ti, V, and Zn; Switzerland, ICP-AES/ICP-MS: Co, V, and Zn).Local variations (=within sampling sites) in element concentrations were estimated, based upon in-site multiple sampling and analysis of Pleurozium schreberi species in The Netherlands. Element concentrations in moss species were compared in linear correlations, both in unweighted and weighted fits, with weighing factors based on the local variation data. Weighted fits were shown to generally improve the calibration characteristics, as tracked by X (2) calculations.The calibration data suggest the presence of previously unnoticed outliers in element concentrations. The absence of further information, however, may prescribe the use of all data in comparison procedures. These results indicate that interlaboratory analysis of replicate samples and the use of dedicated certified reference materials may help solving problems in the analysis of the sample series.For several of the considered interspecies comparisons, weighted calibrations could be based on significant correlations (P=0.05). Actual use, however, will remain arbitrarily decided upon, and may be based on decisions as to what to accept with respect to the levels of uncertainty in the calibration parameters. Furthermore, the use of calibrations in extrapolation modes is greatly restricted by the necessary reservations in geographically larger-scaled applications.