ALBERT

Notes: Soluble hydrocarbon and dissolved oxygen (DO) in a shallow aquifer beneath a field site were characterized by sampling ground water at 42 monitoring wells. Results from 10 sampling periods over three years showed a significant reduction in total benzene mass with time in ground water. These reduction and leakage rates from sources were determined from material balance and nonlinear least-squares analyses. The natural attenuation rate was calculated to be 0.95%/day. Spatial relationships between DO and total benzene, toluene, and xylene (BTX) were shown to be strongly correlated by statistical analyses and solute transport modeling. In addition, laboratory microcosm biodegradation experiments were performed to determine possible threshold limits for aromatic hydrocarbon oxidation under varying levels of dissolved oxygen. The results were remarkably consistent with field data on the presence of high or low levels of BTX and DO in several monitoring well-water samples.

Notes: Development of a numerical algorithm to analyze slug test data is described. This type of test is very popular for aquifer testing, primarily because of its simplicity. Many such tests are performed to estimate the hydraulic conductivity values of ground-water-bearing formations. Those values in turn are used to calculate pore-water velocities. The algorithm was coded and successfully tested for a hypothetical data set. It has also been applied at a number of field locations. One such application is presented.

Notes: Abstract Prosthetic devices undergo corrosion processes after implantation including the release of certain amounts of metal ions into the adjacent tissues. On reaching the bloodstream, a systemic influence of those ions may be envisaged. Cell adhesion molecules (CAMs) are recognized as an essential component of the mechanisms of endothelial damage. To study the influence of selected heavy metals on human umbilical vein endothelial cells (HUVEC) EIA methods were used to evaluate cellular expression of E-selectin, ICAM-1, VCAM-1 and GMP-140 under the influence of high (cytotoxic) very low (non-cytotoxic) concentrations of Zn, Ni, Co and Cr. The de novo synthesis of CAMs was studied with the help of mRNA analysis. Intermediate voltage immuno electron-microscopical imaging was performed to detect the localization on the cell surface of the adhesion molecules E-selectin and ICAM-1 under the influence of cytokines, which represent important factors in inflammatory processes. Very low concentrations of metal ions, which gave no significant influence on cell morphology, elicited a significant expression of CAMs on endothelial cells in vitro. Thus, for example, zinc, nickel and cobalt ions in concentrations of 1×10-9 M increased the expression of endothelial E-selectin, compared to the control after a 5 h incubation. Similar findings were established for zinc, nickel and cobalt ions also with regard to ICAM-1, VCAM-1 and GMP-140. Northern blot analysis gave an increased ELAM-1 and ICAM-1 mRNA expression after incubation with high concentrations of zinc and nickel ions. The results should draw attention to possible effects of very low concentrations, which are released during processes of metal corrosion on prosthetic devices.

Notes: Deleterious effects of dental alloys, especially those of dental amalgams, have become an important issue in current discussions on biomaterials. Cytotoxicity and further related risks of amalgams are discussed in a controversial way in the literature without leading to a final conclusion. There is still a need for basic clinical and pre-clinical research, especially with respect to the wide distribution of dental amalgams. Standardized methods of cytotoxicity testing have been established by the ISO. It was the aim of the present study to detect and compare possible cytotoxic effects of dental amalgams and alternative non-amalgam alloys in vitro. According to the ISO standards, direct contact tests and extract dilution tests were performed using the cell lines HeLa and L-929 as well as primary isolated human fibroblasts, a relevant cell type of the human gingiva. For direct contact tests the samples were fixed on thermanox discs. Zn and Ni-chloride in defined molar concentrations were used as positive controls in the extract dilution tests, while copper was the positive control in the direct contact tests. The tested amalgam was a Non-Gamma-2 amalgam. For extract dilution tests sixteen extraction dilutions were performed. The different cell types were incubated with the extracts in 96-well microtitre plates. MTT-testing was performed to evaluate the effects on cellular metabolism. The BrdU labelling index was determined with the help of EIA methods to analyse the effects of the extracts on the cellular proliferation at DNA synthesis level. The morphological status of the cells seeded on the materials (direct contact test) were studied with the help of light microscopy. No cytotoxic effects of formerly extracted dental amalgam was found, although fresh amalgam elicited a significant cytotoxic effect, in general the non-amalgams have to be regarded as non cytotoxic. The negative control and the non-amalgams elicited no measurable cytotoxicity in the indirect contact assays, independent of the number of extraction dilutions. This applied to all cell types studied. The tested amalgam also gave a significant cytotoxic effect in the MTT-assays, while in addition a significant reduction of BrdU incorporation after incubation with the extracts of the first dilution series, compared to the silicone control was found. The effects were reduced after an incubation with the extracts of the higher dilution series. It is suggested by the presented results that amalgams might have cytotoxic effects, especially when being freshly applied. The cytotoxic effects were no longer detectable after extraction procedures. Nevertheless, a negative effect around such amalgams must be considered. The insights provided by the present studies might be helpful for a rational choice of dental materials.

Notes: This investigation forms part of a study on the suitability of human omentum mesothelial cells (HOMES) as an alternative to endothelial cells (EC) for seeding vascular grafts. Isolated HOMES were grown in primary culture and characterized by their morphology (light microscopy and scanning electron microscopy (SEM)), as well as by fluorescence-activated cell sorting (FACS) and immunocytochemistry. The latter two methods showed cells which were positive for smooth muscle-type actin and cytokeratin, but negative for factor VIII-related antigen. HOMES were grown to confluence on glass with or without a fibronectin coating. Controlled shear stress was applied for up to 30 min using a plate and cone rheometer at 20 dynes/cm2. These dynamic culture conditions led to loss of only occasional cells. The most marked alterations seen on SEM were some cell elongation, marked raising of the nucleus and loss of luminal cytoplasmic microvilli. Time-lapse video microscopy revealed that shear stress also increased the spreading capacity of some cells. Similar experiments with venous endothelial cells gave a shearing off of a confluent monolayer. This investigation shows the marked shear-stress resistance of HOMES, a pre-requisite for their use to seed vascular prostheses.

Notes: Abstract Determination of potential cytotoxicity is a central issue in current biocompatibility testing standards such as ISO and ASTM. Most of these tests do not assess biocompatibility of a biomaterial with regard to cell function. This study was aimed at screening a number of potential parameters that could be included in assessment of cell functional aspects of biocompatibility. Human umbilical vein endothelial cells (HUVEC) were seeded directly on titanium, NiCr alloy, CoCr alloy, PMMA, PE, PU, PVC, and silicone, or were exposed to the material extracts. Cytotoxicity was assessed for these materials through MTT conversion, crystal violet protein determination and Ki67 expression. In addition, expression of the cell adhesion molecules E-selectin, cadherin-5 and PECAM, as well as of the adhesion-associated proteins fibronectin and vinculin (focal adhesions), was determined by immunocytochemistry and western blotting. Cytotoxicity was not detected with the material extracts. Cells were able to adhere to bare metals, but not polymers. Fibronectin preadsorption resulted in adhesion and spreading also on the polymers. Cells were able to establish cell–cell contacts and focal adhesions. Western blotting, in combination with differential detergent extraction, indicated that linkage of cell–cell adhesion markers to the cytoskeleton may be used as an additional parameter relevant to cell function.

Notes: Abstract The past two decades have witnessed a revolution in our understanding of chemical processes in living organisms. This is mainly a result of the massive advances in the fields of cell and molecular biology. These techniques are highly relevant to the biomaterials sector, as they offer the scientist the possibility to better understand the mechanisms involved in the interactions between cells and a material surface—a prerequisite for the rational development of medical devices with optimal biocompatibility. The purpose of the present article is to explain the rationale of the cell and molecular biological approach to biomaterial research and to present typical examples from the authors’ laboratory, as well as from the literature, to illustrate its application. Important aspects of interfacial biology, including the underlying biological mechanisms and methodology, are presented. Of the latter the combination of morphological techniques with methods of cell and molecular biology as well as molecular genetics (so-called “combinative techniques”) are particularly useful. The applicability of this approach is illustrated from a study on the pathomechanisms of metal ion-induced inflammation. In addition, the approach is essential to the development of targeted intervention strategies, as for example in the luminal surface modification of vascular prostheses to permit endothelial cell seeding.

Notes: Abstract The increasing number of patients requiring prosthetic substitution of segments of the vascular system strongly supports the need to optimize a relevant, standardized testing panel for new materials designed for synthetic vascular prostheses. The ISO gives the standard requirements for testing biomaterials provided for implantation. Our primary interest was the establishment of a reliable in vitro panel as a useful and relevant screening system for vascular implant devices to evaluate blood/device interactions under flow conditions. The aim of the present study was to evaluate influences of different flow conditions on blood cell–biomaterial interactions with special emphasis on the interactions of human granulocytes (PMN) and polymeric surfaces. PMN were isolated and vital cells were quantified by flow cytometrical analysis directly before, as well as immediately after the experiments. The viscosity of the final cellular suspension was analysed by using a computerized cone-plate rheometer. As reference materials we used FEP-teflon, PVC-DEHD, PU, PP and PE. Dacron and ePTFE synthetic vascular protheses were tested in a comparative way to those references. The adhesion processes were observed over a period of 40 minutes under arterial (shear stress 0.74 Pa) and venous (shear stress 0.16 Pa) flow conditions in a parallel plate flow chamber system under highly standardized conditions and laminar flow. The cells were observed with the help of inverse light microscopy. Cell behaviour was recorded and analysed in both analogue (video) and digital (imaging system) modes. Samples of the cell suspensions were obtained at regular time intervals and analysed by enzyme linked immuno sorbent assay (ELISA) to quantify LTB4 release. Irrespective of the material, approximately 3 to 4 times more PMN adhered to the biomaterial surfaces under venous flow conditions compared to the arterial. Shear intensity did not influence the running order of biomaterials with respect to cell numbers. This response in descending order at the end of the experiments was as follows: PU, PVC-DEHD, PP, PE and ePTFE. The biochemical analyses indicate that in the system used only a weak effect on LTB4 release induced by the different materials could be determined. A significant effect caused by flow conditions was not observed. Further experiments, both static as well as dynamic, must be performed for multiple, relevant parameters of haemocompatibility, for potential biomaterials as well as those currently in use in vascular prostheses.

Notes: Abstract A quantitative method to assess cell proliferation is one essential prerequisite for testing biomaterial cytocompatibility in vitro. Currently used methods, e.g. bromodeoxyuridine incorporation, show serious disadvantages concerning either sensitivity, specificity or handling. A new enzyme linked immunosorbent assay (ELISA) system for the quantification of cell proliferation based on detection of the Ki-67 protein is described. This protein has turned out to be strictly correlated with the active parts of the cell cycle but to be absent in G0. The measurement of Ki-67 expression by different human cell types, e.g. endothelial cells and HeLa cells, was evaluated in order to answer the question of whether the data obtained using the Ki-67 ELISA method correlate with the proliferation measured with flow cytometrical DNA analysis and microscopical evaluation. Methods currently used for the evaluation of cell proliferation were compared to the new Ki-67 ELISA method. In addition, the functionality of adherent endothelial cells, and the viability and morphology of the cells were investigated. Cells were treated with standard culture medium with or without the transcription inhibitor, actinomycin D, or growth factors, e.g. endothelial cell growth factor (ECGF), and were exposed to metal ion standard solutions. These solutions were in a cytotoxic–non-cytotoxic range. Ki-67 ELISA was found to be a reliable quantitative method to assess proliferation of cultured human cells in vitro. It has advantages over methods that are currently being used. It is easy to perform and corresponds to the requirements for a test to be selected for biomaterial testing according to ISO standard 10 993.