Biomedical subjects

A Bruinink

Publications and source records attributed to A Bruinink.

At least 19 recordsLinked to original sources

PubMed2004

Investigating cell-material interactions by monitoring and analysing cell migration.

Cell-material interactions can on one hand be characterised by assessing the functional state and or shape of the cells at one or different discrete periods of time, on the other hand by observing cell migration and spreading behaviour. The object of this study was to investigate the migration behaviour of fluorescently labelled cells, and to evaluate the software analysing this migration. In the present study, the behaviour of fibroblasts cells on differently structured surfaces was taken as example. In the first step, the influence of seven different lipophilic dyes (Dil, DiO, DiA, DiD, DiR, PKH2 and PKH26) on cell performance was determined taking biochemical parameters as indices. In the second step, the fluorescence characteristics of these dyes were compared regarding their applicability. In the third step, migration behaviour of Dil-labelled fibroblastic cells on plane and grooved surfaces were monitored and analysed using specific software. Our data suggest that most of the dyes have optimal characteristics for studying cell-cell interactions. Cell migration behaviour regarding migration direction and cell spreading was different on plane and grooved surfaces. It could be shown that computer-based image analysis represents a practical, quick and objective tool to quantify exactly cell migration behaviour.

3T3 Cells↗

PubMed2004

Effects of serum and serum heat-inactivation on human bone derived osteoblast progenitor cells.

Generally, heat inactivated foetal calf serum (FCS) containing media are used for the cultivation of animal and human cells. The role of serum source and serum treatment on the behaviour of cells has long been neglected. The present study was performed to investigate the effects of serum heat inactivation and serum source on trabecular bone derived progenitor cells (HBC). Furthermore, it was investigated in how far these reactions differed from those seen in bone marrow derived mesenchymal progenitor cells (HBMC) cultures. We found that HBC cultures performed differently in the presence of FCS and HS with or without heat inactivation. The reactions similar to some degree those observed in HBMC cultures. The implications of the results on cell-implant surface interaction studies are discussed.

Aged↗

PubMed2002

Short-term assay for the identification of neurotoxic compounds and their liver derived stable metabolites.

The release of stable neurotoxic metabolites from liver after metabolic activation of xenobiotics can be investigated in cultures of primary rat hepatocytes as metabolic activation system and embryonic chicken brain cell cultures as target. It was shown that adult rat hepatocyte cell cultures are a reliable bioactivating system for xenobiotics such as cyclophosphamide and isophenphos resulting in the release of stable metabolites into the supernatant. The cultured embryonic chicken brain cells were able to discriminate between the toxicity of parent drugs and its metabolite(s) and between metabolites with an unspecific cytotoxic activity (cyclophosphamide) and metabolites with a high potential to damage specific nerve cell population(s) (isophenphos). The observed neurotoxicity of the isophenphos metabolites is not an acute effect but induced only after a prolonged exposure period. The present study provides evidence that the subsequent use of hepatocytes and brain cell cultures has the potential to be used as an in vitro screening system for the identification of specific and non-specific neurotoxic compounds. Solely stable metabolites are taken into account, since in the in vivo situation only stable metabolites have a change to reach the nervous system. Our data suggest that the present approach is able to discriminate (a) between cell-type and organ specific toxicity, (b) between neurotoxicity derived after long-term or acute exposure, and (c) between the neurotoxicity induced by the native test compound or stable metabolites.

Animals↗

PubMed2001

Grooves affect primary bone marrow but not osteoblastic MC3T3-E1 cell cultures.

To elucidate the influence of microtextures on bone cell performance, primary adult rat bone marrow cells (RBMC) and osteoblastic MC3T3-E1 cells were cultured on tissue culture pretreated plates to which grooves at different density were applied. RBMC cells were found to be significantly affected by grooves in the substratum in contrast to osteoblastic MC3T3-E1 cells, taking culture morphology, total cell number, cell mass, and cell activity (MTT-dehydrogenase), parameter for differentiation of osteoblast progenitor cells into (pre-)osteoblasts (alkalinephosphatase activity, ALP) and tartrate-resistant acid phosphatase (TRAP) activity as indices. TRAP is located in lysosomes and secretory granules mainly although not solely in osteoclasts. By applying grooves to and/or by chemical treatment of unpretreated pure polysterene plates it could be concluded that the effects on RBMC cells were evoked not only by the presence of grooves but also by the surface chemistry of the grooved and ungrooved surface areas.

3T3 Cells↗

PubMed2001

The stiffness of bone marrow cell-knit composites is increased during mechanical load.

A novel device for mechanical stimulation of primary adult rat bone marrow cells cultured on three-dimensional knitted textiles has been prototyped. A method has been developed ensuring a well-defined, high-density, and reproducible cell seeding on the knitted fabric. After culturing for 18-52 days the cell-knit composites were subjected to uniaxial 2% stretching and relaxation. The frequency was altered between 0.1 Hz (196 min, loading phase) and 0.01 Hz (360 min, resting phase). Identically treated knits without cells exhibited a slight stiffness reduction, whereas the stiffness of knits with cells increased from cycle to cycle. The stiffness increase was found to depend on the duration of the culture period before mechanical loading. Our data suggest that the extracellular matrix deposited by the cells on the knit and intact microtubuli of living cells cause the observed stiffness increase. In comparison to the unstrained static cell-knit composites cell proliferation and bone cell differentiation were reduced by the mechanical load.

Animals↗

PubMed2000

Titanium containing amorphous hydrogenated carbon films (a-C: H/Ti): surface analysis and evaluation of cellular reactions using bone marrow cell cultures in vitro.

Amorphous hydrogenated carbon (a-C : H) coatings, also called diamond-like carbon (DLC), have many properties required for a protective coating material in biomedical applications. The purpose of this study is to evaluate a new surface coating for bone-related implants by combining the hardness and inertness of a-C : H films with the biological acceptance of titanium. For this purpose, different amounts of titanium were incorporated into a-C : H films by a combined radio frequency (rf) and magnetron sputtering set-up. The X-ray photoelectron spectroscopy (XPS) of air-exposed a-C : H/titanium (a-C : H/Ti) films revealed that the films were composed of TiO2 and TiC embedded in and connected to an a-C : H matrix. Cell culture tests using primary adult rat bone marrow cell cultures (BMC) were performed to determine effects on cell number and on osteoblast and osteoclast differentiation. By adding titanium to the carbon matrix, cellular reactions such as increased proliferation and reduced osteoclast-like cell activity could be obtained, while these reactions were not seen on pure a-C : H films and on glass control samples. In summary, a-C : H/Ti could be a valuable coating for bone implants, by supporting bone cell proliferation while reducing osteoclast-like cell activation.

Analysis of Variance↗

PubMed2000

Feasibility study of an online toxicological sensor based on the optical waveguide technique.

Morphological properties of the cells often change as an early response to the presence of a pharmacologically acting toxic substance [Etcheverry, S.B., Crans, D.C., Keramidas, A.D., Cortizo, A.M., Arch. Biochem. Biophys. 338 (1997) 7-14]. Recently it has been shown that living animal cell adhesion and spreading can be monitored online and quantitatively via the interaction of the cells with the evanescent electromagnetic field present at the surface of an optical waveguide [Ramsden, J.J., Li, S.Y., Heinzle, E., Prinosil, J.E. Cytometry 19 (1995) 97-102]. In the present study, optical waveguide lightmode spectroscopy (OWLS) and confocal laser scanning microscopy (CLSM), which provides information about the shape of the cells at the surface, were compared under identical experimental conditions. This allowed for the correlation between the cell-shape information from CLSM and the cell-surface interaction measurements from OWLS. The proposed design of the microsystem sensor involves the establishment of a cell layer on the surface of the waveguide and the subsequent online measurement of the morphological response of the cells to various toxic substances. In the present study, the setup was evaluated using cells from an osteoblastic MC 3T3-E1 cell line, and sodium hypochlorite was used as model toxic substance. Comparing the OWLS signal to the morphological response measured by CLSM reveals that OWLS is effective in monitoring not only cell attachment and spreading but also the cellular response to toxic compounds (i.e. by means of change in cell morphology). For the model toxin, the OWLS measurements indicate that, at concentrations above 0.01%, the cells exhibit a clearly discernable morphological effect (i.e. a decrease in average cell contact area). Thus, the potential of an on-line sensor based on OWLS to applications in toxicology, pharmacy and biocompatibility was demonstrated.

3T3 Cells↗

PubMed2000

[Growth of human respiratory epithelium on collagen foil].

BACKGROUND: Clinical application of bioartificial tracheal prosthesis must still be regarded as an experimental concept because restoration of a functional respiratory epithelium outlining the prosthesis is still not possible. Tissue engineering as a relatively new biotechnological discipline may offer new methods in expanding differentiated respiratory epithelium in vitro. In this study we compare two different cell and tissue culture procedures for growing human nasal mucosa on commercially available collagen foil. MATERIAL AND METHODS: Harvested specimens of human nasal mucosa (n = 6, 4 x 4 cm) were placed on collagen foil and incubated as tissue cultures for 4, 6 and 8 weeks. A suspension of enzymatically dispersed nasal epithelium seeded on collagen foil (5 x 10(5) cells) served as control. Cell growth and ciliary beat were monitored through an inverted microscope with Hoffman's modulation contrast and video set-up. Histological examination was performed after 4, 6 and 8 weeks. RESULTS: In the tissue cultures, the collagen foil was initially covered with fibroblasts growing from the mucosa specimen before epithelial cells spread out. The epithelial layer showed mostly ciliated cells which developed metachronous ciliary beat after 4 weeks in vitro. Ciliary activity was observed until the end of the experiments in 8 weeks. New cells on the suspension cultures were mesenchymal and did not exhibit any ciliary activity. CONCLUSIONS: Mucosa specimens seem to be more appropriate for tissue engineering of respiratory epithelium than cell suspensions from nasal epithelium. Collagen foil as tissue scaffold initiates epithelial-mesenchymal interaction and may play an important role in epithelial differentiation of new respiratory epithelium.

Biomedical Engineering↗

PubMed1999

[Biomaterials, human tolerance and integration].

Biomaterials and related process engineering in order to obtain optimal surface and structural biocompatibility of implants and devices are presented. Vital-avital composites for tissue engineering, cell culture models, porous ceramics and degradable polymers are introduced as examples. Emphasis is laid on the conversion of basic research results into clinical applications and on the exchange of technologies from the non-medical into the medical field and vice versa.

Animals↗

PubMed1998

Growth inhibitory factor and zinc affect neural cell cultures in a tissue specific manner.

Deficiency of neuronal growth inhibitory factor (GIF) and abnormalities in zinc homeostasis have been suggested to play a role in the neuropathogenesis of Alzheimer's disease. We report here that embryonic chick cerebral cell cultures zinc and copper containing GIF in the presence of marmoset hippocampal extract reduces significantly and concentration dependently mitochondrial succinate dehydrogenase activity (MTT) and cell mass. In contrast, no indications could be found that GIF affected neural retina cell cultures. Our results suggest that the observed effects of GIF are not elicited by zinc.

Animals↗

PubMed1998

Differences in neurotoxic effects of ochratoxin A, ochracin and ochratoxin-alpha in vitro.

The mycotoxin ochratoxin A (OTA) is a chlorinated dihydroisocoumarin derivative connected through an amide-bond to L-phenylalanine. In a previous study we could show that competition with L-phenylalanine-dependent processes does not play a role in OTA neurotoxicity. To test whether the isocoumarin part is responsible for the neurotoxic effects, we determined in the present study the effects of the hydrolysis product of OTA, ochratoxin-alpha (OTalpha), and of ochracin on embryonic chick brain cell cultures. In addition, we investigated the interaction between OTA and ochracin regarding the neurotoxic effects. We report here that OTalpha did not affect brain cell cultures at concentrations up to 15 microM. With the exception of a small (20%) but significant reduction in cell culture, cellular protein at concentrations above 0.3 microM, in our cell cultures' cell function, as defined by neutral red uptake and MTT-dehydrogenase activity, was only reduced by high OTalpha concentrations (1 mM). Addition of 0.1 microM OTA increased ochracin cytotoxicity as defined by latter parameters. No effects on cell culture NF68kD content could be detected. The results are discussed with regard to the existence of an OTA target interaction binding site.

Animals↗

PubMed1998

In vitro techniques for the assessment of neurotoxicity.

Risk assessment is a process often divided into the following steps: a) hazard identification, b) dose-response assessment, c) exposure assessment, and d) risk characterization. Regulatory toxicity studies usually are aimed at providing data for the first two steps. Human case reports, environmental research, and in vitro studies may also be used to identify or to further characterize a toxic hazard. In this report the strengths and limitations of in vitro techniques are discussed in light of their usefulness to identify neurotoxic hazards, as well as for the subsequent dose-response assessment. Because of the complexity of the nervous system, multiple functions of individual cells, and our limited knowledge of biochemical processes involved in neurotoxicity, it is not known how well any in vitro system would recapitulate the in vivo system. Thus, it would be difficult to design an in vitro test battery to replace in vivo test systems. In vitro systems are well suited to the study of biological processes in a more isolated context and have been most successfully used to elucidate mechanisms of toxicity, identify target cells of neurotoxicity, and delineate the development and intricate cellular changes induced by neurotoxicants. Both biochemical and morphological end points can be used, but many of the end points used can be altered by pharmacological actions as well as toxicity. Therefore, for many of these end points it is difficult or impossible to set a criterion that allows one to differentiate between a pharmacological and a neurotoxic effect. For the process of risk assessment such a discrimination is central. Therefore, end points used to determine potential neurotoxicity of a compound have to be carefully selected and evaluated with respect to their potential to discriminate between an adverse neurotoxic effect and a pharmacologic effect. It is obvious that for in vitro neurotoxicity studies the primary end points that can be used are those affected through specific mechanisms of neurotoxicity. For example, in vitro systems may be useful for certain structurally defined compounds and mechanisms of toxicity, such as organophosphorus compounds and delayed neuropathy, for which target cells and the biochemical processes involved in the neurotoxicity are well known. For other compounds and the different types of neurotoxicity, a mechanism of toxicity needs to be identified first. Once identified, by either in vivo or in vitro methods, a system can be developed to detect and to evaluate predictive ability for the type of in vivo neurotoxicity produced. Therefore, in vitro tests have their greatest potential in providing information on basic mechanistic processes in order to refine specific experimental questions to be addressed in the whole animal.

Animals↗

PubMed1997

Reduction of ochratoxin A toxicity by heat-induced epimerization. In vitro effects of ochratoxins on embryonic chick meningeal and other cell cultures.

The widespread contamination of food by mycotoxins may present a serious hazard to human and animal health. The present study was designed to determine the toxic potential of three structurally related ochratoxins: ochratoxin A (OTA), ochratoxin B (OTB) and the heat-induced 3S-epimer of OTA (3S-OTA) recently discovered in roasted coffee and human serum. The toxicity was determined using serum-free cell cultures of embryonic chick meningeal fibroblasts, taking the effects on mitochondrial and lysosomal activity and culture protein content as an index for toxicity. OTA, OTB and 3S-OTA were toxic. However, the concentration necessary to induce comparable effects were nearly 19- and 10-fold higher for OTB and 3S-OTA, respectively, than those for OTA. In a next step the sensitivity of serum-free cell cultures of embryonic chick neural retina and brain were compared in relation to meningeal cell cultures. In the present study, no indications for differences in sensitivity could be detected. Furthermore, our study suggest that the OTA-induced toxic effects are not due to the inhibition by OTA of phenylalanine-tRNA synthetase.

Animals↗

PubMed1997

The neurotoxic effects of ochratoxin-A are reduced by protein binding but are not affected by l-phenylalanine.

Recent in vivo investigations indicate that the mycotoxin ochratoxin A (OTA) is a neurotoxicant during prenatal stages. In line with in vivo data, in our embryonic chick brain and neural retina cell cultures the markers for neuritic outgrowth and differentiation (NF68 and 160 kDa, MAP2 and MAP5) were especially negatively affected. In vivo OTA is nearly completely bound to serum constituents. In our culture system binding of OTA to BSA evoked a significant shift of the concentration-effect relationships in meningeal and brain cell cultures. As a result of the albumin binding the OTA IC5 and IC50 values of all parameters increased by nearly the same value (about 15-fold in brain and 32-fold in meningeal cell cultures). One of the mechanisms responsible for OTA toxicity is thought to be the competitive inhibition versus Phe of Phe-dependent enzymes. Therefore, in addition, we investigated the effects of l-phenylalanine (Phe) and its influence on OTA toxicity in brain and neural retina cell cultures. Phe itself was found to differently affect brain and neural retina cell cultures. However, in both cultures OTA toxicity is not diminished by Phe. Therefore, our data indicate that at least in our cultures competition with Phe-dependent processes does not play a role in OTA toxicity.

Animals↗

PubMed1996

Neurotrophic effects of transferrin on embryonic chick brain and neural retinal cell cultures.

The viability and differentiation promoting effects of various transferrins [iron-saturated (holo) and iron-depleted (apo) human and chick ovo (conalbumin)-transferrins, and bovine apo-transferrin] were studied, using serum-free, flat-sedimented cell cultures of embryonic chick brain and neural retina. The effects of transferrin (Tf) on the cell cultures depended on the type of Tf used and the parameter measured. Significant differences between brain and neural retina cultures in the effects of apo-ovoTf and iron [supplemented as ammonium-iron (III) citrate] were detected. Maximal levels of mitochondrial activity were observed in the presence of 2 mg/l apo-ovoTf in neural retina cell cultures. In brain cell cultures, 40 mg ovoTf/l were needed to achieve maximal levels. In brain, but not in neural, retina cell cultures ovoTf and optimal concentrations of Fe3+ exhibited similar effects on biochemical parameters of cell function and differentiation. Although, in the absence of ovoTf, neuronal outgrowth on areas not covered by glial cells was inhibited in both cell cultures, the differences were more prominent in neural retina cell cultures. Our data strongly suggest that Tf plays a key role in processes not connected directly with its iron transport capability.

Animals↗

PubMed1994

Neurotoxic effects of aluminium on embryonic chick brain cultures.

Toxic damage of brain cells by aluminium (Al) is discussed as a possible factor in the development of neurodegenerative disorders in humans. To investigate neurotoxic effects of Al, serum-free cultures of mechanically dissociated embryonic chick (stage 28-29) forebrain, brain stem and optic tectum, and for comparison meningeal cells, were treated with Al (0-1000 microM) for 7 days. Effects of Al on cell viability (lysosomal and mitochondrial activity) and differentiation (synthesis of cell-specific proteins) were found to the brain area specific with the highest sensitivity observed in optic tectum. No inhibiting effects on cell viability could be observed in cultures of forebrain and meninges in the concentration range tested. In all three brain tissue cultures, threshold levels for the reduction of cell differentiation parameters were found at lower concentrations [concentration resulting in a 50% decrease (IC50) > 180 microM] than for the inhibition of cell viability (IC50 > 280 microM), indicating a specific toxic potential of Al for cytoskeletal alterations. The culture levels of nerve cell-specific markers microtubule-associated protein type 2 (the most sensitive parameter) and the 68-kDa neurofilament were inhibited at lower concentrations (IC50 180-630 microM) than the astrocyte-specific glial fibrillary acidic protein (IC50 700-approximately 1000 microM), demonstrating a particularly high sensitivity of neurons in comparison to astrocytes. Based on these differences in Al sensitivity observed for different cell markers in the various brain tissue cultures, the in vitro system used in the present study proved to be a suitable model to assess brain area and cell type-specific neurotoxic effects of Al.

Aluminum↗

PubMed1993

Dopamine D2 receptors are unevenly distributed in the rat hippocampus and are modulated differently than in striatum.

The characteristics of dopamine (DA) D2 receptors were studied in rat hippocampus and for comparison in striatum using the [3H]spiperone radioreceptor assay in vitro. D2 receptors exhibit a bimodal distribution along the length of the hippocampus. Heterogeneity also exists in the transverse axis with high densities in the molecular layers of the subiculum and CA1 plus CA2. As in the striatum, D2 receptors in both dorsal and ventral hippocampus display high and low affinity states for agonists, but all three regions differ with regard to the percentage of receptors in these states. The modulation of these affinity states by GTP, Mg2+ and the iron-chelator, 1,1-O-phenanthroline, differs markedly between dorsal and ventral hippocampus, and between these regions and the striatum. A new model for the modulation of the affinity of D2 receptors for agonists is presented. These data suggest the presence of regional differences in the composition of the D2 receptor-regulatory protein complex.

Animals↗

PubMed1993

Effects of cisplatin and ORG.2766 in chick embryonic brain cell cultures.

The neurotoxicity of cisplatin and ORG.2766, both separately and in combination, was investigated using serum-free chick embryonic brain cell cultures. At low cisplatin concentrations glial cells were affected more than nerve cells. The onset of cisplatin toxicity was delayed, the major effects only being seen after the treatment was finished and when no free cisplatin was present in the culture medium. The data further indicate that the area under the graph of free cisplatin concentration in the culture medium against time (AUC) is a measure of cisplatin toxicity. The AUC of free cisplatin in the culture medium which causes a 50% reduction in the expression of glial fibrillary associated protein (GFAP) was similar to blood AUC values in humans known to induce neurotoxic effects in around 80% of patients. ORG.2766 at very high concentrations increased lysosomal activity, as measured by neutral red uptake, and the expression of GFAP. ORG.2766 did not influence the toxicity of cisplatin in culture.

Adrenocorticotropic Hormone↗