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  • 1
    ISSN: 1434-4726
    Keywords: Tonsillectomy ; Oral mucosa ; Keratinocytes ; Cell Culture ; Keratin pattern
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Tonsillectomy tissue can be used as a routine source for cultures of oropharyngeal keratinocytes. In so doing, a peritonsillar strip of unaltered mucosa was dissected in the upper submucosa. Subsequent trypsinization yielded 7.0 \+- 3.4 × 106 keratinocytes per bilateral tonsillectomy. Keratinocyte attachment and growth in primary culture were promoted by sublethally irradiated 3T3 murine fibroblasts. Three subcultures could be performed without a feeder layer and were characterized by a population doubling time of 4.5 days during log growth phase. Electrophoretic and immunoblot analysis of the third subculture revealed a strong expression of keratin pairs 5/14 and 6/16 as well as keratins 7 and 19, whereas keratins 8/18 were expressed less intensely. The lowest intensity was found for keratin 13, which is known to be indicative of the differentiated mucosa. The culture technique thus provides an easily available in vitro model for morphological and functional studies on the epithelial compartment of human oropharyngeal mucosa.
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  • 2
    ISSN: 1433-0458
    Keywords: Schlüsselwörter Epithelisierung ; Gewebeersatz ; Biokompatibilität ; Screening ; In vitro ; Keywords Epithelization ; Tissue replacement ; Biocompatibility ; Screening ; In vitro
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Abstract Successful use of non-biologic implants in reconstructive head and neck surgery is dependent on tissue compatibility and epithelization. This is true not only for epithelial cells, but also for mesenchymatic cells. Therefore we tested several substrates with human fibroblasts or keratinocytes from the oral mucosa in cell culture. In tissue culture keratinocyte outgrowth from small mucosal flaps onto the surface was observed. Preparations were evaluated by histology and scanning electron microscopy. Cellulose-ester, polyvinylidene-difluoride and polyglactin developed monolayers of fibroblasts and keratinocytes in cell cultures. In tissue culture mucosal flaps showed good adherence to the surface of these materials and a fine outgrowth of keratinocytes. Expanded polytetrafluor-ethylene (ePTFE) was partially covered by a layer of fibroblasts and keratinocytes in cell culture, but cell adherence was not sufficient. In tissue culture the mucosal flaps failed to attach on ePTFE. These results illustrate that the mesenchymatic and epithelial component of cell and tissue cultures show different qualities of cellular adherence and growth on the surface of non-biologic implants. We propose our method for the development of an in-vitro model for the epithelization of non-biologic implantation materials.
    Notes: Zusammenfassung Der Einsatz nicht-biologischer Implantate in der plastisch-rekonstruktiven HNO-Chirurgie wird durch deren Gewebeverträglichkeit und Epithelisierbarkeit bestimmt. Dabei ist nicht nur die Akzeptanz des Implantats durch Epithelzellen, sondern ebenfalls durch mesenchymale Zellen wichtig. Als In vitro-Epithelisierungsmodell wurden 4 verschiedene, nicht-biologische Materialien in der Zellkultur einerseits mit oralen Keratinozyten (Mukosakorrelat) und andererseits mit Fibroblasten aus Temporalisfaszie (Submukosakorrelat) besiedelt. Zusätzlich wurde in der Gewebekultur das Auswachsen von Epithelzellen aus oralen Schleimhautstücken (”outgrowth culture”) beurteilt. Die Präparate wurden histologisch und/oder rasterelektronenmikroskopisch untersucht. Zellulosemischester, Polyvinylidendifluorid sowie Polyglactin wurden in Zell- und Gewebekulturen von beiden Zelltypen gut besiedelt. Dagegen zeigten expandierte Polytetrafluorethylen-Streifen (ePTFE) von 0,1 mm Dicke in der Zellkultur einen eingeschränkt adhärenten, einschichtigen Fibroblastenbewuchs. Keratinozyten hatten eine mangelhafte und Schleimhautstücke gar keine Adhäsion. Die vorliegende Untersuchung zeigt, daß sowohl die Zell- als auch die Gewebekultur sehr gut geeignet sind, in einem in vitro Modell nicht-biologische Gewebeersatzmaterialien zellulär zu besiedeln und qualitative Unterschiede bezüglich der Zelladhäsion und Bewachsung darzustellen. Unser In vitro-Modell, welches sowohl eine epitheliale als auch eine mesenchymale Komponente humaner Zellen berücksichtigt, stellt die erfolgreiche Entwicklung einer Screeninguntersuchung zur Epithelisierbarkeit nicht-biologischer Materialien in Aussicht.
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  • 3
    ISSN: 1432-069X
    Keywords: Epidermal cells ; Cell proliferation ; Crystal violet ; Cellular protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The application of a simple, rapid, and inexpensive colorimetric growth assay was tested for human epidermal cells subcultured in uncoated plastic dishes. Cell layers were incubated with a crystal violet (CV) solution (0.2% with ethanol 2% in 0.5 M Tris-Cl buffer, pH 7.8) for 10 min at room temperature. After rinsing with 0.5 M Tris-Cl (pH 7.8) the cell layer was dried and decolorized with a sodium-dodecylsulfate solution (0.5% with ethanol 50% in 0.5 M Tris-Cl, pH 7.8) for 60 min at 37°C. The extinction of the supernatant was read at the absorption maximum of 586 nm. The protein content of attached cells as classical parameter for quantifying cell growth was strongly related to CV extinction with a correlation coefficient of r=0.98. Furthermore, the subcellular protein binding qualities of CV were analyzed. The water-soluble protein fraction of cultured epidermal cells was separated by sodium-dodecylsulfate polyacrylamide gel electrophoresis and stained with CV. We found a staining pattern which was qualitatively very similar to that of Coomassie blue, however less intense. Keratin electrophoresis revealed an affinity of CV to the 48, 50, and 56 kD cytokeratins. In conslusion, this CV assay is a reliable and simple method for the monitoring of epidermal cell growth in cultures.
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  • 4
    ISSN: 1432-069X
    Keywords: Keratinocytes ; Dithranol ; Lactate dehydrogenase (LDH) ; Cytotoxicity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary HaCaT cells, a rapidly multiplying human keratinocyte line, were tested for their sensitivity to antipsoriatic dithranol with regard to classical proliferation parameters and for the drug's action on the plasma membrane integrity by the dose- and time-dependent release of cytosolic lactate dehydrogenase (LDH). In the case of 3H thymidine as well as 14C amino acid incorporation the 50% inhibition concentration (IC50) was 0.2 ΜM dithranol 24 h after initial exposure to the drug. For protein content of attached cells the IC50 proved to be〉3.0 ΜM. Using 0.3, 1.0 and 3.0 ΜM dithranol, significant (p〈0.05) dose dependent LDH release of 0.866±0.387, 1.842±1.127 and 2.938±1.635 mU per hour and cm2 confluent culture area was measured between the 5th and the 24th hour, compared to an acetone control of 0.504±0.299 mU/ h×cm2. Between the 2nd and the 4th hour as well as from the 25th to the 48th hour and the 49th to the 72nd hour the LDH release after dithranol treatment did not exceed the control value. In accordance with these findings dose-dependent morphological signs of cell injury were detected by phase contrast microscopy beyond the 4th hour. The data reveal that: HaCaT cells are a very sensitive target for the antiproliferative action of dithranol; the drug causes considerable plasma membrane damage even at concentrations as low as 0.3 ΜM; and this membrane damage becomes evident after a latency of at least 4 h and for a limited period of up to 24 h.
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  • 5
    ISSN: 1432-069X
    Keywords: Human keratinocytes ; Dithranol (anthralin) ; Tolerance ; Tachyphylaxis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The hyperproliferative human keratinocyte line (HaCaT) was tested for dithranol tolerance (tachyphylaxis) at the cellular level. At day 4 after seeding, keratinocytes were treated with 0.3 or 1.0 ΜM dithranol. Data were compared with those from experiments including additional pretreatments with 0.3 ΜM at day 3, or at days 1 and 3. Protein content, DNA synthesis and protein synthesis (incorporation of 3H-thymidine and 14C-amino acids per protein) were determined at 24, 48 and 72 h after the last drug exposure. Protein content of attached cells decreased in relation to dose and frequency of treatments. Inhibition of DNA and protein synthesis (38.2% and 32.3%, respectively) also occurred 24 h after a single treatment with 0.3 ΜM dithranol, but was only 18.4% and 9.1% after pretreatment twice with 0.3 ΜM dithranol. This tolerance reaction in vitro, after repeated dithranol exposure of human keratinocytes, may be explained by a selective loss of drug-sensitive cells.
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  • 6
    ISSN: 1432-069X
    Keywords: Tiflucarbine ; Protein kinase C ; Keratinocyte proliferation ; Reactive oxygen species ; Psoriasis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Various studies have suggested that calmodulin (CaM) is involved in the pathophysiology of psoriasis. Protein kinase C (PKC) is also accepted as playing a regulatory role in cell proliferation as well as in inflammatory processes. Therefore, we investigated the effects of the known CaM antagonist tiflucarbine (BAY/TVX P 4495) on two cellular systems related to the major clinical symptoms of psoriasis: proliferation of cultured human keratinocytes (HaCaT cell line) and release of reactive oxygen species (ROS) from human polymorphonuclear leukocytes (PMNL). Tiflucarbine inhibited both cellular responses in a dose dependent manner. Furthermore, tiflucarbine directly affected PKC, and may thus be considered to be a dual PKC/CaM antagonist with putative antipsoriatic activity. The effects of tiflucarbine on the different parameters were compared with those of the structurally unrelated dual PKC/CaM inhibitor W-7 and those of the potent PKC inhibitor staurosporine. The potencies of all three compounds were found to be in the same range as their PKC-inhibiting potency. Our data indicate that PKC, rather than CaM, may play a regulatory role in the release of ROS as well as in keratinocyte proliferation. Therefore, inhibition of PKC in general might have a therapeutic benefit in psoriasis.
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  • 7
    ISSN: 1432-069X
    Keywords: Anthralin ; Stability ; Phospholipids ; Liposomes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
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  • 8
    ISSN: 1432-069X
    Keywords: Arylhydrocarbon-hydroxylase (AHH) ; Epidermal cells ; Cell proliferation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Cytochrome P-450-dependent arylhydrocarbon-hydroxylase (AHH) activity and inducibility by benzanthracene (BA) was measured in cultured guinea pig and human epidermal cells. Basal AHH-activity (AHHb) in guinea pig epidermal cells was much higher than in human epidermal cells. AHHb in guinea pig epidermal cells was directly related to the labeling index and decreased to the original level between the 5th and 7th day of cell culturing. On the other hand, the induction-ratio of AHH reached its maximum level when the number of cells began to rise (proliferation phase) and remained high at day 7 of the cell culture. These results suggest a cell growth dependent activity and inducibility of carcinogen-metabolizing enzymes, such as AHH, in isolated epidermal cells.
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  • 9
    ISSN: 1432-069X
    Keywords: Epidermal cells ; Lipid fluidity ; Diphenylhexatriene ; Fluorescence polarization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
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  • 10
    ISSN: 1365-2133
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The in vitro effects of liposomes on HaCaT human keratinocytes were studied with regard to their uptake, lipid fluidity and proliferation of the cells. Oligolamellar liposomes, prepared from soya bean phospholipids. had a mean size of 150nm and consisted predominantly of phosphatidylcholine (83%) and phosphatidylethanolamine (10%) and the fatty acids comprised mainly linoleic acid (66%) or other unsaturated fatty acids. After 6 and 24 h of incubation with 1 and 0–1% w/v of liposomal lipids, phase-contrast microscopy revealed marked cytoplasmic vacuolization of the cells. Keratinocytes treated with the liposomes contained aggregations of multilaminated lipid material without delimiting cell membranes. The cellular lipid fluidity (reciprocal of diphenylhexatriene fluorescence polarization P-value) correlated with liposomal concentration and incubation time. A significant elevation of lipid fluidity (P 〈 0.05) was observed with 1 and 0.1%) liposomes after 1 h of incubation (81.8 ± 4.7 and 95.7 ± 1.2% of control P value) and for 0.01% liposomes after 3 h (96.2 ± 1.5%). Maximum fluidity occurred after 48 h of exposure to 1% liposomes (42.1 ± 3.1%). Exposure to liposomal lipids for 24 and 48 h resulted in suppressed cell proliferation with 50% inhibition concentrations (IC50), being 0.06% for incorporation of [3H]-thymidine, 0.08% for [14C]-amino-acid incorporation and 〉 1% for protein content per well after 24 h of exposure. The cells were able to proliferate and lipid fluidity returned to normal within 7 days following discontinuation of incubation with liposomal lipids.
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