Bernard N. Bachra

Effects of glucocorticosteroids on primary human skin fibroblasts

SummaryVarious glucocorticosteroids were added to logarithmically growing cultures of primary human skin fibroblasts and of mouse L929 fibroblasts. These steroids inhibited proliferation of the human fibroblasts at concentrations which fall in a range expected to occur during the topical treatment of skin disorders. In terms of the concentrations required for the inhibition hydrocortisone was least and clobetasol-17-propionate most effective. All other steroids studied (hydrocortisone-17-butyrate, triamcinolone acetonide, betamethasone-17-valerate and hydrocortisone-21-acetate) showed medium effectiveness. Fluorination as such may not enhance the inhibitory effect. The inhibition was independent of the source (baby foreskin or adult arm skin) and passage number (7th to 13th or 15th and 16th passage, respectively) of the cells. The possible relationship between the inhibition of cell proliferation by such steroids and their therapeutic effect in psoriasis and their atrophic side effects is discussed.Mouse L929 fibroblasts were affected at 103–104-fold lower steroid concentrations and the range of the effective concentrations was 104–105 times as wide as that for the primary human skin fibroblasts. It was concluded that these mouse fibroblasts are a poor model system for the study of in vivo effects of glucocorticosteroids in man.ZusammenfassungEine Anzahl Glucocorticosteroide wurden logarithmisch wachsenden Kulturen primärer menschlicher Hautfibroblasten und L929 Mäuse-Fibroblasten hinzugefügt.Im Konzentrationsbereich, der während der lokalen Behandlung von Hauterkrankungen in der Haut zu erwarten ist, wurde die Vermehrung menschlicher Fibroblasten gehemmt. Die zur Hemmung erforderliche Konzentration war für Hydrocortison am höchsten und für Clobetasol-17-propionat am niedrigsten. Die Hemmung durch die anderen Steroide (Hydrocortison-17-butyrat, Triamcinolon-acetonid, Betamethason-17-valerat und Hydrocortison-21-acetat) erfolgte im Zwischenbereich. Die Hemmung wurde durch Fluorinierung an sich nicht gesteigert und war unabhängig von der Herkunft dieser Fibroblasten (Baby-Vorhaut oder Armhaut Erwachsenen) und von der Zahl der Passagen (bzw. 17–23 oder 15–16). Mögliche Beziehungen wurden diskutiert zwischen der Hemmung der Zellvermehrung durch diese Steroide einerseits und ihren therapeutischen Effekten bei Psoriasis anderseits, sowie ihre atrophischen Seiteneffekte.L929 Mäuse-Fibroblasten wurden schon bei 103–104fach niedrigeren Steroidkonzentrationen merklich beeinflußt und der wirksame Bereich war 104–105fach breiter als für primäre menschliche Hautfibroblasten. Daraus folgt, daß diese Mäuse-Fibroblasten ein ungeeignetes Modellsystem für Untersuchungen der in vivo-Effekte von Glucocorticosteroide beim Menschen bilden.

Effects of glucocorticosteroids on cultured human skin fibroblasts—IV: Specific decrease in the synthesis of collagen but no effect on its hydroxylation

Abstract Confluent cultures of normal baby foreskin fibroblasts were exposed for 6 days to hydrocortisone-17-butyrate (5 μg/ml)or to clobetasol-17-propionate (1 or μg/ml). On day 5 [ 3 H]proline was added to the cultures and on day 6 both medium and cell layer were analyzed for [ 3 H]protein and protein-bound [ 3 H]hydroxyproline. The synthesis of labeled protein was little affected, while that of labeled collagenous protein was greatly depressed, as compared to that of the control cells. This depression occurred for the collagenous protein present in the cell layer as well as for that released into the growth medium. This effect was not accompanied by a decrease in cellular prolyl hydroxylase activity or in collagen proline hydroxylation.

Calcification in vitro of demineralized bone matrix. Electron microscopic and chemical aspects.

Collagen was prepared from compact sheep bone by decalcification with EDTA and from rat tail tendons by acetic acid extraction and reconstitution with NaCl. The deposition of apatite in sheep bone collagen in a metastable calcification solution was studied chemically and by electron microscopy. The bone collagen was shown to be a good nucleation catalyst for mineral deposition, while rat tail collagen was a poor catalyst. Mineral deposition in bone collagen occured in two separate kinetic phases, a rapid phase of nucleation and crystal growth, giving rise to small calcified islands, and a second slow phase, ascribed to growth in regions not involving the catalytic sites. This second phase of mineral deposition is considered to be the result of impaired ion diffusion through the closely-aligned collagen fibrils, thus leaving large areas of the collagen free of mineral even though the buffer remains highly supersaturated. Electron micrographs suggested that the catalytic sites might be in some relationship to the 640 A periodicity of collagen, but a role for non-collagenous material bound to the collagen has not been excluded.

Effects of glucocorticosteroids on cultured human skin fibroblasts. III. Transient inhibition of cell proliferation in the early growth stages and reduced susceptibility in later growth stages.

An immediate depression of the rate of cell proliferation occurred upon addition of glucocorticosteroids to cultures of human skin fibroblasts in the early growth stages. A reduced sensitivity or even insensitivity of the fibroblasts to growth inhibition inhibition was found upon the addition of the steroids at later stages of cell growth, when the cell density has increased. The inhibition in the early growth stages is transient and is most pronounced if the cultured medium is not renewed. This transient inhibition is not due to the development of steroid-resistant cell lines, and resembles the effect called tachyphylaxis, as is also observed in vasoconstriction tests.

Effects of glucocorticosteroids on cultured human skin fibroblasts

SummaryAs previously found, the glucocorticosteroid clobetasol-17-propionate inhibits cell proliferation during the early growth stage of normal baby foreskin fibroblasts and collagen synthesis in confluent cultures of these cells. The degree of inhibition of cell proliferation decreases with increasing cell density and, moreover, is transient.The anabolic steroids nandrolone and nandrolone-phenyl-propionate have similar effects on these cells. Likewise the magnitude of the inhibition is dose-dependent.When present together the two types of drug do not act in an additive manner. Even at low concentrations the anabolic steroids abolish the inhibitory effect of the glucocorticosteroid on cell proliferation. Furthermore, in this case only the inhibitory effect of the glucocorticosteroid on collagen synthesis is found and there is no further increase in this effect due to the presence of the anabolic steroids.Our results imply that the use of low concentrations of anabolic steroids combined with glucocorticosteroids in topical application to the skin may abolish some of the undesirable side effects of the glucocorticosteroids.ZusammenfassungWie schon früher festgestellt wurde, hemmt das Glucocorticosteroid Clobetasol-17-propionate die Zellvermehrung während der frühen Wachstumsstadien der normalen Babyvorhautfibroblasten und die Kollagen-Synthese in konfluenten Kulturen der Zellen. Der Hemmungsgrad der Zellvermehrung sinkt mit zunehmender Zelldichte und ist außerdem von kurzer Dauer.Die anabolen Steroide Nandrolone und Nandrolone-phenyl-propionate haben ähnliche Effekte auf diese Zellen. Ebenso ist die Hemmungsgröße konzentrationsabhängig.Bei Anwendung beider Medikamente haben diese keine additive Wirkung. Schon bei niedrigen Konzentrationen unterdrücken anabole Steroide den Hemmeffekt der Glucocorticosteroide auf die Zellvermehrung. Weiter wird in diesem Fall nur der Hemmeffekt der Glucocorticosteroide auf die Kollagen-Synthese gefunden, und keine weitere Steigerung des Effekts in Anwesenheit von anabolen Steroiden.Unsere Resultate lassen vermuten, daß der Gebrauch niedriger Konzentrationen von anabolen Steroiden in Kombination mit Glucocorticosteroiden bei (äußerlicher) Applikation auf die Haut einige der unerwünschten Nebenwirkungen aufheben könnte.

The effect of tetracycline and oxytetracycline on the formation of biological apatite

Recently, an interesting paper was published in this Journal by Kaiti la (1971) which strongly suggested tha t tetracycline affects the formation of biological apat i te in cultured mouse embryo bones (ulnae and radii) by a direct effect on the formation of the mineral phase. At a tetracycline concentration of only 10 ~g/ml the uptake of calcium was significantly lowered. Although clearly suggesting a direct effect on mineral formation, these results do not differentiate an effect on the nucleation and crystal growth of the mineral as such from an effect on the biological phenomena which might precede these physicochemical processes. For a number of years we have been interested in the mechanism of action of inhibitors of biological calcification. We have studied the effects of added ions of Mg and F (Bachra et al., 1965 ; Bachra and Fischer, 1969), Sr (Baehra and Fischer, 1969) and of various polyvalent metals (Bachra and Van Harskamp, 1970). The addition of Mg, F and Sr ions at relatively low concentrations resulted in the inhibition of nucleation. At relatively higher concentrations crystal growth was also inhibited. On the contrary, many of the polyvalent metal ions destabilized the otherwise metastable calcification buffer. I t was shown further tha t Mg, F and Sr ions did not have a specific effect on the catalytic action of demineralized bone matr ix in apat i te nucleation in a model system different from the effect on the nucleation of apat i te as such.