G. Neupert

Adhesion, spreading and growth of animal cells on the surface of glass ceramic Ap40--a contribution to the cell compatibility of dental permanent hard tissue implants.

Cells of different cell lines on the surface of glass ceramic Ap40 responded in the manner of reversible vacuolization in the cytoplasm. Whereas the eluate of bulk glass ceramic did not produce toxic effect on cell populations in vitro, the eluate of pulverized glass ceramic Ap40 caused a toxic reaction, the effect being dependent on concentration and incubation time. Although cell attachment, spreading and proliferation of fibroblast-like and epithel-like cells on glass ceramic surfaces are delayed when compared to glass and polystyrene the cells are adhering to a confluent monolayer.

Cytokeratin expression in experimental murine rhabdomyosarcomas. Intermediate filament pattern in original tumors, allotransplants, cell culture and re-established tumors from cell culture

Soft tissue sarcomas were induced by 20-methylcholanthrene in NMRI-mice. The tumors were characterized as rhabdomyosarcomas by light and electron microscopy as well as immunohistochemistry (vimentin, desmin and myoglobin expression). Cytokeratins could be demonstrated by a panel of different poly- and monoclonal antibodies in original rhabdomyosarcomas, their allotransplants and the re-established tumors from cell culture in nude mice. The cytokeratin positive tumor cells were arranged in small clusters and/or haphazardly single dispersed in the rhabdomyosarcomas. By means of monoclonal antibodies cytokeratins No. 8 and No. 19 could be evidenced and cytokeratin No. 18 could be made probably. Behind the background of cytokeratin expression in developing fetal cross striated muscle cells our findings are discussed as a reminiscence of embryonal muscle development in these tumors. The significance of cytokeratin expression in rhabdomyosarcomas for diagnostic histopathology is emphasized.

Immunohistochemical localization of cytochrome P450 1A1 in precision-cut rat liver slices after in vitro exposure to β-naphthoflavone

Mono- and polyclonal antibodies have been used to study the localization and distribution of cytochrome P450 1A1 (CYP1A1) in cultured precision-cut liver slices with various immunohistochemical methods. Neither in non-incubated slices nor in slices incubated in the absence of beta-naphthoflavone (BNF) for 24 hrs was CYP1A1 immunohistochemically detectable. After incubation in the presence of BNF (25 microM), however, CYP1A1 was well visible in parenchymal and biliary epithelial cells. CYP1A1 was not evenly distributed, but was localized predominantly in hepatocyte layers near the surfaces of the slices. This distribution could be due to the preferential uptake of BNF by outer cell layers or due to functional changes of inner cells. Together with results obtained with other methods (e.g. RT-PCR) this investigation also demonstrates that precision-cut liver slices are a useful tool for the detection of in vitro induction of CYP1A1.

Experimentally induced metastases of malignant fibrous histiocytomas xenotransplanted into nude mice from an established sarcoma cell line (RFS)

An experimental approach for inducing metastases from xenotransplanted malignant fibrous histiocytomas in nude mice is presented. Malignant fibrous histiocytomas were generated by inoculation of an established cell line (RFS) in the back of 16 nude mice. 7 nude mice were subjected to diminution operations carried out twice and three times, respectively, 6 out of these animals developed metastases, whereas no metastases were found in the inoperated control group (9 mice). The metastasis formation is explained by sarcoma heterogeneity, some factors which might contribute to the phenomenon of tumor dissemination are discussed.

Growth of fibroblasts and epithelioid cells on a new machinable bioactive glass ceramic in comparison with non-reactive materials

In this study scanning electron microscopy (SEM) was used to investigate the substrate attachment, cell spreading and growth properties of fibroblasts and epithelioid cells on a newly developed machinable bioactive glass ceramic. The morphogenetic reactions of cell cultured on bioactive ceramic were compared with those of identical cells cultured on non-reactive glass and vitreous carbon. The adhesion, subsequent spreading and the growth appearance of fibroblasts and epithelioid cells on the surface of machinable glass ceramics are similar to those on nonreactive materials. Our findings concerning the behaviour of cell populations in vitro on the surface of a new machinable glass ceramic allow the conclusion that this implant material is adhesive for cells and biocompatible.

Growth inhibition and morphological changes caused by indomethacin in fibroblasts in vitro.

In a definite range of concentrations indomethacin inhibits the multiplication of fibroblasts in vitro without influencing their viability. The effect of lethal doses of indomethacin on the fine structure of normal and transformed fibroblasts was investigated by light and electron microscopy. The cells grown in flat monolayers became roundish and, varying in sequence, small zeiotic blebs were bulging at the external cell membrane. Beside coarse vesicular transformation of the endoplasmatic reticulum and extension of the perinuclear spaces ballooning of the mitochondria occurred. After breaking up of the vesicles of the endoplasmatic reticulum loosening of the cytoplasmic ground substance associated with enlargement of the hyaline plasma margins was observed in further progress of the experiment. The internal structures of the mitochondria were completely disorganized. In the ultrastructure of the fibroblasts altered by lethal doses of indomethacin no specific sites of drug action could be identified.

The glycosaminoglycan metabolism of chondrocyte monolayer cultures under normal and pathological conditions. A methodic study.

Chondrocyte cultures may serve as a model in investigating changes of the cartilage metabolism. Adherent chondrocytes in vitro maintain polygonal morphology at high cell density in the primary and secondary culture. Collagen type II is only clearly detected in multilayered or nodular areas. The differentiation of the chondrocytes is also indicated by a low HA concentration of the cultural medium. It depends on high cell density, a low number of subcultures and their duration. However, the medium GAG of chondrocyte cultures does not exactly mirror the state of cell differentiation but can partly be used to check it. Subcultures of chondrocytes on small cover slides (minicultures) are used to determine proteoglycan synthesis and degradation for 48 h each. Both synthesis and degradation of cell-associated GAG or proteoglycans, resp., follow similar complex kinetics. The half lives of sulfated GAG or proteoglycans are initially 10 h (T-1 for O-6 h of chase), later 39 h or 95 h (T-2 for 6-48 h of chase). Conditioned medium of casein-elicited rat peritoneal macrophages reduce the sulfate incorporation into chondrocyte proteoglycans and their degradation rates increase. In the additional presence of E. coli endotoxin (0.5 microgram/ml) the synthesis of proteoglycans is only little affected; the degradation rate is stronger increased. To peritoneal macrophages of rats manifold pretreated with BCG and perhaps desensitized, LPS is added in vitro. Conditioned medium of these MP does not affect the chondrocyte proteoglycan synthesis but enhances the degradation rates in a concentration-dependent manner. Thus it can be demonstrated that chondrocyte monolayer miniscale cultures may serve to elucidate changes in the proteoglycan synthesis and different degradative steps.

Characterization of established epithelioid cell lines derived from rat liver: expression of cytokeratin filaments

A number of epithelioid cell lines were established from livers of fetal and neonatal rats. The clear-epithelial cells of these lines are non-neoplastically altered, clonogenic, nearly-diploid and showed no hepatocyte-like properties. The distribution and organization of intermediate filaments were analysed by indirect immunofluorescence and immunoperoxidase techniques using polyclonal and monoclonal antibodies against cytokeratins and vimentin. The different keratin antibodies also react strongly with intermediate filaments of bile duct cells on frozen sections of fetal and neonatal liver. Several subcultures of all established cell lines contained cell populations which reacted positively with antibodies against prekeratin and broad-range cytokeratins in a different manner, but not with antibodies against cytokeratin No. 19 of the catalogue of MOLL. The intermediate filaments are arranged in predominantly stained pericellular rings as well as in fine filamentous networks more evenly distributed throughout the cytoplasm. In early passages almost all cells were found positive for pre- and cytokeratins whereas in later subcultures the number of positive cells decreases and the expression of cytokeratin polypeptides varies considerably as seen with the different antibodies. All cells of the different cell lines show a well developed fine filamentous vimentin network extending throughout the whole cell up to the outer cell margin. Our findings support the concept that the clear epithelioid cells in established cell lines are of epithelial nature but they are not derived from biliary epithelium, and therefore probably immature parenchymal liver cells.

Myofibroblasts in connective tissue capsules around implanted dental materials

For testing the biocompatibility of dental materials in an experimental animal model the capsular tissues around the implanted dental materials were examined light and electron microscopically. The exudative inflammation and fibrosis at different times after implantation were thought to be the indicators of the degree of the compatibility. Starting from the knowledge of the occurrence of myofibroblasts in reparative granulation tissue we looked for such cells in the capsular tissues. Myofibroblasts were especially found at three weeks after onset of the experiments. Structural peculiarities of this fibroblast modulation are emphasized. Myofibroblasts are considered as an essential link between the early exudative reaction and the late fiber formation in tissue reactions after mechanical injury.

Acid mucopolysaccharides in fibroblast cultures: 3.Influence of lactate on the MPS-distribution pattern and 35S-sulfate incorporation at different cell densities

Summary The influence of 100 mg % L-lactate in the culture media of monolayers of embryonic rat fibroblasts was studied at p H 7.4.Cell proliferation, amount of total MPS, MPS-distribution pattern, 35 S-sulfate incorporation (relative synthesis rate, specific activity, MPS-metabolism) were determined in their dependence on cell density.