The effects of collagen fragments on the extracellular matrix metabolism of bovine and human chondrocytes.

Cartilage matrix degradation generates collagen type II fragments. The objective of this study is to explore the possibility that these collagen fragments may be part of an endogenous metabolic feedback. Initially, collagen fragments were extracted from normal or osteoarthritic cartilage, as part of a matrix fragment preparation. Later, collagen fragments were generated by digestion of bovine collagen type II with bacterial collagenase (col2f). These fragments were added to cultures of isolated chondrocytes (bovine and human) and cartilage explants (human). In a dose-dependent manner, col2f caused inhibition of cell attachment to collagen, inhibition of collagen synthesis, and induction of matrix degradation. In addition, when col2f were added to human cartilage explants, an induction of gelatinase activity was detected in the media. These data sets present first evidence that degradation products of collagen may be directly involved in the regulation of cartilage homeostasis.

A microarray enzyme-linked immunosorbent assay for autoimmune diagnostics.

In order to quantify autoantibodies in the sera of patients with autoimmune disease, we have created a microarray-based immunoassay that allows the simultaneous analysis of 18 known autoantigens. The microarrays contain serial dilutions of the various antigens, thereby allowing accurate determination of autoantibody titer using minimal amounts of serum. The assay is very sensitive and highly specific: as little as 40 fg of a known protein standard can be detected with little or no cross-reactivity to nonspecific proteins. The signal intensities observed from serial dilutions of immobilized antigen correlate well with serial dilutions of autoimmune sera. Miniaturized and highly parallelized immunoassays like these will reduce costs by decreasing reagent consumption and improve efficiency by greatly increasing the number of assays that can be performed with a single serum sample. This system will significantly facilitate and accelerate the diagnostics of autoimmune diseases and can be adapted easily to any other kind of immunoassay.

Organotypic culture system of chicken retina.

Analysis of developmental mechanisms during neuroembryogenesis, evaluation of toxicological effects and testing of neuroprotheses rely to an increasing extent on in vivo-like in vitro models. We have developed a novel organotypic culture system of the chick retina. Tissue slices of embryonic retinae were immobilized on glass coverslips by a fibrin clot and permanently rotated between the gas and medium phase, resulting in regular formation and the maintenance of the retinal cytoarchitecture. Selection of embryonic stage, slice thickness and specimen processing were optimized for culturing. Scanning electron microscopy revealed degradation during increasing culture periods of the fibrin clot, which was used for initial immobilization of explants on glass coverslips. Simultaneously, retinal cells became exposed on the tissue surface. Even after several weeks in vitro, formation and maintenance of plexiform and nuclear layers was evident as revealed by two specific monoclonal antibodies. Immunocytochemistry employing two additional photoreceptor- and radial Müller-antibodies indicated differentiation of neuronal and glial cells specific for the retina. The organotypic culture system promises to facilitate developmental studies of retinal development. Quantitative evaluation of Na(+)-channel blocker mexiletine impact on the histogenesis of retinal explants proved the organotypic culture system to be a valuable tool also for neurotoxicological investigations.

Induction of cAMP-dependent protein kinase A activity in human skin fibroblasts and rat osteoblasts by extremely low-frequency electromagnetic fields.

Sinusoidal extremely low-frequency electromagnetic fields (ELF-EMF; 7-8 mT, 20 Hz) have already been shown to inhibit proliferation and to accelerate terminal differentiation of human skin fibroblasts in vitro. In order to elucidate the underlying processes of signal transduction, we analysed the activity of cAMP-dependent protein kinase (PKA). EMF exposure for 60 min resulted in an increased PKA activity in human skin fibroblasts (2-fold) and rat embryonic osteoblasts (1.7-fold). Long-term exposure for up to 7 days with a constant 1 h-on/1 h-off EMF exposure rhythm indicated a transient stimulation of PKA activity during the first two exposure rhythms followed by a decrease to the baseline levels of sham-exposed controls. Based on these results, we postulate that a modulation of proliferation and differentiation processes in cells of mesenchymal origin is triggered by an immediate and transient EMF-induced increase in PKA activity.

Induction of intracellular calcium oscillations in human skin fibroblast populations by sinusoidal extremely low-frequency magnetic fields (20 Hz, 8 mT) is dependent on the differentiation state of the single cell.

Experiments were performed to analyze whether short-term exposure to a sinusoidal extremely low-frequency electromagnetic field (20 Hz, 8 mT) can alter the dynamics of intracellular calcium in diploid human skin fibroblasts. In heterogeneous fibroblast populations, about 30% of the cells responded with a change in the oscillation activity of intracellular calcium within 40 min. It was demonstrated at the level of the single cell that the responsiveness of fibroblast populations to extremely low-frequency electromagnetic fields depends on the specific differentiation state of the exposed cell. The data obtained clearly indicate that mitotic progenitor fibroblasts respond with an enhancement of the dynamics of calcium, whereas in postmitotic fibrocytes a reduction of the dynamics was observed when the cells were co-stimulated with suboptimal concentrations of platelet-derived growth factor. Thus data from our laboratory on terminal differentiation induced by extremely low-frequency electromagnetic fields may be correlated with changes in the dynamics of Ca2+ reported here.

Stimulation of protein kinase A activity and induced terminal differentiation of human skin fibroblasts in culture by low-frequency electromagnetic fields.

In the present study experiments are described, which indicate that the exposure of normal human skin fibroblasts (HSFs) to a low-frequency electromagnetic field of 20 Hz and 7-8 mT does induce terminal differentiation within 14 days of daily exposure of 12 h. As demonstrated by the analysis of the expression level of the proto-oncogene c-myc, the induction of terminal differentiation of progenitor fibroblasts to postmitotic fibrocytes does most likely not involve changes in the c-myc protein. As one possible candidate being involved in the ELF-EMF-mediated inhibition of fibroblast growth and the subsequent induction of terminal differentiation the cyclic AMP-dependent protein kinase A (PKA) could be characterised. Thus, the data presented clearly indicate that the specific EMF field of 20 Hz and 7-8 mT significantly interfere with regulatory processes of fibroblast proliferation and differentiation.

Bioelectronic noses: a status report. Part II.

The present state of the art to record or to mimic electronically the human senses of olfaction and taste is characterized. In this part II, strategies are outlined to utilize chemical and biological structures with their different complexities which serve as sensor elements in (bio-) electronic noses. Finally a survey is given on the computer-science aspects of odor recognition based on these elements.

A novel organotypic long-term culture of the rat hippocampus on substrate-integrated multielectrode arrays.

Spatiotemporally coordinated activity of neural networks is crucial for brain functioning. To understand the basis of physiological information processing and pathological states, simultaneous multisite long-term recording is a prerequisite. In a multidisciplinary approach we developed a novel system of organotypically cultured rat hippocampal slices on a planar 60-microelectrode array (MEA). This biohybrid system allowed cultivation for 4 weeks. Methods known from semiconductor production were employed to fabricate and characterize the MEA. Simultaneous extracellular recording of local field potentials (LFPs) and spike activity at 60 sites under sterile conditions allowed the analysis of network activity with high spatiotemporal resolution. To our knowledge this is the first realization of hippocampus cultured organotypically on multi-microelectrode arrays for simultaneous recording and electrical stimulation. This biohybrid system promises to become a powerful tool for drug discovery and for the analysis of neural networks, of synaptic plasticity, and of pathophysiological conditions such as ischemia and epilepsy.

Analysis of 3H-proline-labeled protein by rapid filtration in multiwell plates for the study of collagen metabolism.

Quantitative and qualitative analysis of cell cultures labeled with 3H-proline for monitoring collagen synthesis is time-consuming and occasionally generates large quantities of radioactive waste. This present work describes the application of a microwell filtration system for the analysis of collagen metabolism in chondrocytes. It is based on trichloroacetic acid (TCA) precipitation of 3H-proline-labeled proteins onto polyvinylidene difluoride membranes fitted in a 96-well plate and subsequent analysis of precipitated proteins by liquid scintillation counting, amino acid analysis and sodium dodecyl sulfate (SDS) gel electrophoresis. This method allows for the initial processing of 96 samples within 2 h, has high sensitivity and accuracy (linearity > or = 200 cpm/sample) for quantitative measurements and a capacity of up to 10 micrograms collagen/microwell. The ratio of the radioactive protein collected by this filtration assay compared to that collected by molecular sieve chromatography on Sephadex G-25 was linear over a broad range, indicating full compatibility of data and a high reproducibility for both assay systems. The quality of protein separation by SDS-polyacrylamide gel electrophoresis (PAGE) from samples obtained by the filtration assay and by dialysis was virtually identical. These features make the assay particularly suited for pulse-chase experiments and for monitoring protein degradation.

Effects of various hygiene procedures on the surface characteristics of titanium abutments.

The use of cleaning instruments on titanium implants may cause undesired surface alterations. In a qualitative and quantitative assessment of these alterations, 5 titanium implant abutments were treated with a steel curet, a prototype pure titanium curet, an air abrasive polishing system, and an ultrasonic system. Custom-made polymer templates, used to secure the curet to a vertical guide bar and a spring scale to maintain a constant instrument pressure, guaranteed a standardized procedure and reproducible results. The ultrasonic and the air abrasive polishing method were also standardized. Evaluation by scanning electron microscopy (SEM) revealed surface alterations for all instruments and systems except the plastic curet, which did not roughen the surface at all. The confocal laser-scanning microscope allows a 3-dimensional reproduction of these surface alterations and their direct measurement. The profilometric tracing was not sensitive enough to register the minor effects caused by the titanium curet and the air abrasive polishing system. Dimensions of the resulting surface microstructure could be determined with the laser-scanning microscope. Since the influence of such surface defects on the peri-implant tissue reaction is unpredictable, the titanium curet and the air abrasive system can only be recommended with restrictions. The steel curet and the ultrasonic system proved to be totally unsuitable for cleaning titanium implants.

Extracellular recording in neuronal networks with substrate integrated microelectrode arrays.

A photolithographically produced array of 60 substrate-integrated microelectrodes was used for extracellular recording. Neuronal electrical activity was recorded from chicken retinal ganglion cells with or without stimulation by diffuse light. The retina was removed from chicken embryos of embryonic day 14-18. Only cells recorded from day 18 retina would react to photostimulation, increasing their activity when stimulated, corresponding to the developmental time course of photoreceptor differentiation.

A thin film microelectrode array for monitoring extracellular neuronal activity in vitro.

A planar array of microelectrodes has been developed for monitoring the electrical activity of neurons in cell culture. The microelectrode array was tested and characterized using impedance measurements and SEM. To verify the spatial sensitivity of the microelectrodes we used a specially developed simulation device.

Detection of nonvolatile macromolecules in breath. A possible diagnostic tool?

The analysis of parameters in bronchoalveolar extracellular lining secretions has come into greater use in the diagnosis of diseases of the lung and respiratory passages. The bronchoalveolar lavage (BAL) method is thus used for sampling alveolar fluids or bronchial secretions. However, this method is invasive and therefore cannot be routinely employed for probe sampling. Based on the hypothesis that aerosol particles excreted in human breath reflect the composition of the bronchoalveolar extracellular lining fluid, experiments were performed to concentrate and analyze these aerosols directly using a noninvasive technique. Human exhaled air was directed through a set of cool traps and the condensate of 200 to 400 exhalations examined for nonvolatile components, such as proteins. In experiments conducted with volunteers, the amount of proteins in the breath condensate of 8 healthy individuals (of a total of 10) amounted to between 4 micrograms and 1.4 mg. The proteins were separated by two-dimensional polyacrylamide gel electrophoresis (PAGE) and compared to saliva samples of the respective volunteers. The results suggest that the proteins detected in breath originate partially from the naso-oropharyngeal tract and partially from lower regions of the airways. In clinical tests, the exhaled air of 13 patients suffering from various diseases of the respiratory tract was sampled and analyzed by immunoassays for inflammation parameters, such as interleukin-1 beta (IL-1 beta), soluble interleukin-2 receptor protein, light chain (sIL-2R), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha). In these tests, up to 370 pg IL-1 beta, 120 pg TNF-alpha, and 2,159 U sIL-2R per ml were measured in the breath condensate.(ABSTRACT TRUNCATED AT 250 WORDS)

Human endothelial cells are stimulated and vascular smooth muscle cells are inhibited in their proliferation and migration by heparins.

The actions of two natural heparins and a semi synthetic low molecular weight heparin with low anticoagulant activity have been studied on the migration and proliferation of human vascular endothelial and vascular smooth muscle cells in vitro. In a migration assay non irradiated confluent cultures of endothelial cells and smooth muscle cells were "wounded" with a sharp razor blade in such a way, that the migration of individual cells from the wound edge into a region void of cells could be measured. The maximum distance migrated (perpendicularly to the wound edge) three days after wounding, was taken as an index for migratory activity. All tested heparins reduced migration of vascular smooth muscle cells and enhanced migration of vascular endothelial cells in a concentration dependent manner. Cell proliferation was studied in clone culture. All heparins tested were found to inhibit smooth muscle cell growth. The number of clones, as well as the size of single clones, were smaller with increasing concentrations of natural and low molecular weight heparin. The endothelial cells, however, exhibited contrary responses; with increasing concentrations of heparin, the cloning efficiency and the cell number of individual endothelial cell clones increased. This opposite effects of natural heparins and low molecular weight heparin could be of importance in preventing secondary stenosing intimal proliferations after angioplasty, bypass operation and embolectomy or even atherogenesis. Since the low molecular weight heparin has only a low anticoagulatory activity, it may be more appropriate than natural heparins for long term therapy to prevent artery stenoses caused by intimal SMC proliferation.

Expression of smooth muscle myosin in relation to growth kinetics of cultured aortic smooth muscle cells.

The goal of this study is to quantify smooth muscle myosin (SMM) expression at the level of the individual cell and to ascertain whether SMM expression in cultured aortic smooth muscle cells is related to definite growth phases, and whether the initial seeding density affects growth or SMM staining. Rabbit aortic smooth muscle cells (SMCs) were harvested by enzyme digestion of aortic tissue and plated at low (100 cells cm-2), medium (1000 cells cm-2), and high (10,000 cells cm-2) densities. Independent of seeding density, the lag phase lasted 2 to 3 days and, at all three densities, the growth rate during the logarithmic growth phase was almost the same. However, the time, the number of population doubling needed to reach the plateau phase and the cell number in the plateau were influenced by the initial seeding density. Immunofluorescence staining with anti-smooth muscle myosin (ASMM) revealed intensive staining of striated and filamentous patterns in all cells during the lag and early logarithmic growth phases. During the late logarithmic growth phase, two subpopulations of cells appeared, one showing a positive and the other no reaction with SMM antiserum. The lowest relative number of cells which showed positive reactions with SMM antiserum was observed toward the end of the logarithmic growth phase. During the plateau phase, the SMM-positive subpopulation increased, amounting to about 60% of the total number of cells, independent of the seeding density. In terms of absolute numbers, the number of SMM-positive cells increased over the course of 21 days by factors of 13, 72, and 342 for high, medium, and low seeded cultures, respectively. We conclude that a SMC subpopulation can divide without loss of SMM and that some, but not all, cells which lose their SMM may possibly regain it in the postconfluent state.

Cytocontractile structures and proteins of smooth muscle cells during the formation of experimental lesions.

The time course of structural changes in vascular smooth muscle cells (SMC) was investigated during the formation of an experimental lesion in response to balloon injury. We compared the filamentous organization, evaluated by quantitative electron microscopy, with the cellular content of two representative cytocontractile proteins (myosin and tropomyosin) as assessed by immunofluorescence. We found that the changes peak between 7 and 14 days after injury and that they are visible both in the neointima and to a lesser extent in the inner media. While virtually all SMC are of a filament-rich phenotype in the undisturbed media, after balloon injury SMC migrated into the intima and about 90% of these latter cells were either of a organelle-rich or an intermediate phenotype, with the remaining 10% being of the filament-rich phenotype. In the inner media about 40% of cells were either of organelle-rich or intermediate phenotype. In contrast to these profound organizational changes of responding SMC, histochemistry revealed only a slight and probably transient decrease of the cellular content of myosin and tropomyosin at that time point. Twenty-eight days after injury the discrepancies between the content and the organization of cytocontractile proteins became more apparent. While virtually all SMC showed a homogeneous intensive staining with both antibodies, indistinguishable from the media SMC, the organization of cytoplasmic filaments had not totally recovered. Even though this morphological study does not permit conclusions to be drawn on the contractile function of the cells, it shows that both the organization and the content of cytocontractile protein have to be analyzed and compared for SMC changes to be evaluated during the formation of an experimental lesion.

Orientation response of arterial smooth muscle cells to mechanical stimulation.

Arterial smooth muscle cells from rabbit aortic media in primary culture and subculture were grown on hydrophilized and collagen-coated silicone membranes which were then subjected to cyclic and directional stretches and relaxations at a frequency of 60 times/min. The membranes were stretched with various amplitudes ranging from 2% to 20%. Smooth muscle cells on unstretched membranes in the same incubation chamber served as controls. In long-term experiments the stretching and relaxing of the membranes was continued for several days. While the smooth muscle cells grown on unstretched membranes remained in random orientation in all experiments, the cells which underwent mechanical stimulation showed a high degree of orientation. The angle of cell orientation varied in direct relation to the stretching amplitude and became steeper in correlation to the intensity of the mechanical stimulus. The angle of cell orientation was reversible, as preoriented cells changed their orientation when another stretching amplitude was applied. To study the role of cytoskeleton in the process of cell orientation, we examined the behaviour of the intracellular actin filament system. In short-term experiments the smooth muscle cells were exposed for 3 to 12 h to cyclic and directional stretches and relaxations with an amplitude of 10%. We observed a rearrangement of the intracellular actin filament system prior to the orientation of the whole cell bodies. The present study provides evidence that stretching the artery wall by blood pulsation may result in an orientation response of the intracellular actin cytoskeleton and in the orientation of the smooth muscle cells within the media of artery walls.

Co-cultivation of endothelial and smooth muscle cells on opposite sides of a porous membrane.

A co-culture system is here proposed that mimics the topographical situation of the vessel wall in which endothelial cells are separated by a fenestrated elastic lamina from smooth muscle cells. Bovine aortic endothelial cells were grown on one side of a thin microporous membrane and smooth muscle cells were cultivated on the other side. The microporous membrane was inserted in a special frame so that a two-compartment system was created. The membrane may act like the fenestrated internal elastic lamina of arteries in allowing interactions and fluid exchanges between the two cell types through its pores. Membranes were examined both by transmission and scanning electron microscopy to evaluate the morphology of both cell types.

Orientation of cultured arterial smooth muscle cells growing on cyclically stretched substrates.

Arterial smooth muscle cells from rabbit aortic media were grown in first subcultures on hydrophilized and collagen-coated silicone membranes which were then subjected to directional cyclic stretches and relaxations at a frequency of 50 times/min. The membranes were stretched 2, 5 and 10% beyond their resting length. Cells on unstretched and stationary membranes in the same chamber served as controls. The cells which were stretched with an amplitude of 2% remained in random orientation after 14 days of continuously performed cyclic stretching. The cells which were stretched 5% for 12 days orientated at an angle of 61 +/- 9 degrees to the direction of stretching, while the cells which were stretched with an amplitude of 10% for 6 days orientated at an angle of 76 +/- 5 degrees. The cells on the stationary and unstretched membranes remained in random orientation. We were able to confirm that the angle of orientation is reversible, i.e. preorientated cells changed their orientation during application of another stretching amplitude. The results suggest that stretching of the artery wall by blood pulsation may be a factor influencing the orientation of smooth muscle cells within the media of the artery wall and of those smooth muscle cells which proliferate into the subendothelial space after mechanical injury of the endothelium or electrical stimulation of the artery wall. An apparatus is presented which produces cyclic and directional mechanical stimuli similar to those which may occur in the artery wall.