Roger Bassleer

Human chondrocytes in tridimensional culture.

SummaryCartilage was taken from the macroscopically normal part of human femoral heads immediately after orthopedic surgical operations for total prothesis consecuitive to hip arthrosis. After clostridial collagenase digestion and repeated washings, chondrocytes (106 cells) were cultivated in a gyrotory shaker (100 rpm). Under these conditions, cells were kept in suspension and after 3 to 5 d formed a flaky aggregate which, on Day 10, became dense. These chondrocytes were morphologically differentiated: they had a round shape, were situated inside cavities, and were surrounded by a new matrix. Histochemical methods showed the presence of collagen and polysaccharides in cell cytoplasm and in intercellular matrix, and the immunofluorescence method using specific antisera (anticartilage proteoglycans and anti-type II collagen) showed that these two constituts were in tentercellular matrix. The measurement of the amounts of proteoglycans (PG) released into culture media and those present in chondrocyte aggregate (by a specific PG radioimmunoassay) showed a maximum production on Days 3 to 5 of culture, then the production decreased and stabilized (from Day 10 to the end of culture). The observed difference between the amounts of PG in aggregates after 20 d and those after 2 h of culture demonstrated that PG neosynthesis did occur during cultivation. This conclusion was supported by other results obtained by [14C]glucosamine incorporation in chondrocyte aggregates. Moreover, the aggregate fresh weight related to cell number (appreciated by DNA assay) increased significantly with culture duration. Three-dimensional chondrocyte culture represents an interesting model: chondrocytes were differentiated morphologically as well as biosynthetically and synthesized a new cartilage matrix.

Sciatic Nerve Regeneration through Venous or Nervous Grafts in the Rat

This study analyses the interest of isologous venous grafts filled with saline or with Schwann cells versus nerve grafts as guides for regeneration of the sciatic nerve in 35 Wistar rats. Electrophysiological parameters (conduction velocities and distal latencies of motor responses) and the functional index of De Medinacelli were measured several times from 1 month to 1 year after surgery. An histological analysis was performed on 2 control rats and on 3 rats killed 6 or 12 months after surgery: the total number of fibers was counted on a montage photoprint of the whole nerve, and the diameters of axons and the thickness of the myelin sheath were measured on digitized images. With a portion of nerve as guide, the regeneration is faster than with a vein. However, regeneration after 6 months is at least as good with a venous graft filled with Schwann cells, as assessed by electrophysiological, functional, and histological analysis. The addition of Schwann cells in grafted veins allows the nerve to regenerate through longer gaps than previously described (25 vs 15 mm). In order to assess the quality of nerve regeneration, functional, electrophysiological, and histological analysis are complementary.

Screening of cytotoxic activities of Strychnos alkaloids (methods and results)

The potential cytotoxic activities of 46 alkaloids isolated from different Strychnos species were tested on different cancer or normal cells cultured in vitro. The authors used a relatively simple microtest which gives good reproducibility. Most of the active compounds belong to the usambarane skeleton but other structure-activity relationships are being discussed.

Mode of action of cis-dichloro-diammine platinum(II) on mouse ehrlich ascites tumour cells

Abstract Mice bearing an Ehrlich ascites tumour received one intraperitoneal injection of cis -dichlorodiammine platinum (II), cis -Pt (II). The tumour cells were analysed with cytological and quantitative cytochemical methods. Cis -Pt (II) strongly inhibits cell multiplication. When mitosis begins, a block in metaphase usually occurs. This platinum compound blocks the cells in G2 (post-synthesis as to DNA) but, RNA and protein synthesis remaining very active, the cells become giant and contain prominent nucleoli. Later on, in the majority of cases, they slowly degenerate and the ascites tumour tends to disappear. However, tumour growth can sometimes resume.

Effect of selenium compounds on murine B16 melanoma cells and pigmented cloned pB16 cells

The effects of selenium compounds such as sodium selenite, sodium selenate, seleno-Dl-cystine and seleno-Dl-methionine (100 μM and 10 μM) on B16 and pigmented cloned pB16 murine melanoma cells were investigated in vitro. At the tested concentrations, B16 cells showed a greater sensitivity to the toxic effects of sodium selenite and seleno-Dl-cystine than pB16 cells, whereas no decrease of B16 and pB16 cell number was observed after incubation with sodium selenate or seleno-Dl-methionine. Glutathione (GSH) percentages were strongly decreased only by selenite and seleno-Dl-cystine; it was marked more in B16 than in pB16 cells. The pretreatment of B16 cells with a GSH depleting agent (10 μM buthionine-[S,R]-sulfoximine) did not significantly influence the cytotoxic effects of selenite and seleno-Dl-cystine. On both cell populations, GSH preincubation (50 μM) enhanced the cytotoxicity of selenite whereas the survival of seleno-Dl-cystine treated cells was increased. Glutathione peroxidase (GSH-Px) activity in B16 cells was more sensitive than in pB16 cells to the activating effect of selenite, and particularly of seleno-Dl-cystine; however, cell-free controls indicated that activation was mainly due to glutathione reductase. The rate of75Se (as sodium selenite) uptake in both cell populations was maximal within the first hour of incubation, with a preferential accumulation in the cytosol; after 24 h of incubation, the amount of75Se in cytosol and pellet was approximately the same. Gel filtration chromatography of lysed cells after incubation for 6 h with 10 μM75Se-selenite showed that the radioactivity was eluted as two peaks corresponding to low (4–9 kDa) and high (280–320 kDa) molecular weights. Possible toxicological mechanisms are discussed at molecular level. For selenite, a major involvement of GSH is proposed, with production of selenodiglutathione and selenopersulfide, which should be directly responsible of the decrease in cell number, thiol oxidation and protein synthesis inhibition. For selenocystine, an active selenol species (Cy-Se−) is also hypothesized as being responsible for thiol oxidation and mutagenic effects. For both compounds oxygen active species could also be formed; however, a relevant role of GSH-Px was not apparent. The minor sensitivity of pB16 cells to the toxic effects of selenite and seleno-Dl-cystine could be explained by the smaller depletion of GSH induced by those compounds in pB16 cells, a minor formation of selenium active species, the larger amount present of the oxyradical scavenger melanin, the secretion of some mitogenic factor by pB16 cells and/or a greater resistance to autocrine cytotoxic factors.

Effects of peptidic glycosaminoglycans complex on human chondrocytes cultivated in three dimensions

Human chondrocytes from the pelvic joint were cultivated in suspension; under these conditions, after a few days, cells aggregated. These chondrocytes were morphologically differentiated (round shape, situated inside cavities and surrounded by a matrix synthesized during cultivation) and biosynthetically differentiated (synthesis of type II collagen and cartilage proteoglycans (PG) (Bassleer et al. In vitro 22, 115-120, 1986). In this work, we present the metabolic and cellular effects of a peptidic-glycosaminoglycan (P-GAG) complex isolated from calf cartilage and bone marrow. We analyzed the effects of P-GAG on DNA synthesis (appreciated by 3H-thymidine incorporation into DNA), on type II collagen and on PG synthesis analyzed by specific radioimmunoassays. According to its final concentration in culture medium, P-GAG was able to stimulate proliferation or to favor the production of specific components of cartilage matrix, type II collagen and PG.

Cytological and Cytochemical Analysis of the Effects of cis-Dichlorodiamino Platinum (II) on Chick Fibroblasts cultivated in vitro

Chick embryo fibroblasts cultivated in vitro and treated with cis-dichlorodiamino platinum (II) have been analysed by cytological and cytochemical methods. Morphological alterations (in the nucleoli, in the chromatin and in the cytoplasm), inhibition of DNA and RNA syntheses and of cell multiplication have been noted. Under some conditions, cells are blocked just before entering into mitosis (G2 block) and can become polyploid. A high degree of cell degeneration has also been observed.

Antimitotic activity of strychnopentamine, a bisindolic alkaloid

Strychnopentamine has been tested for its cytotoxic and antitumor activities and compared with two other bisindolic alkaloids that possess an usambarane skeleton. The presence of a N-methylpyrrolidine group increases the antimitotic activity of this type of alkaloids.

Effects of ethidium bromide on chick fibroblasts and mouse Ehrlich tumor cells cultivated in vitro. Cytological and cytochemical observations.

Summary Normal chick embryo fibroblasts and Ehrlich ascites tumor cells have been cultivated in vitro in the presence of ethidium bromide (E. B.). Cell growth is strongly inhibited and some cells die. However, the modal cellular volume does not change. The mitochondria are swollen, fragmented and sometimes spherical; many cristae are destroyed and large granular inclusions are present in the matrix. The nucleoli become smaller, denser and the nucleolar constituents are segregated. Some nuclei contain large chromatin blocks. E. B. can be detected in the cell by fluorescence microscopy: it is located in the cytoplasm, in the nucleoli, in the chromatin and inside mitochondria, especially in the intramitochondrial granules. Ethidium bromide can inhibit DNA synthesis and block the cells during DNA synthesis; sometimes, the block happens in G2 (postsynthesis as to DNA). Later on, RNA and protein synthesis can also be inhibited. All these effects are nearly identical in the normal fibroblasts and in the tumor cells.

Matadine, a cytotoxic alkaloid from Strychnos gossweileri

Matadine, a new alkaloid, has been isolated from the root bark of Strychnos gossweileri. Elucidation of its structure is mainly based on 1H and 1D NMR studies. Its cytotoxic activity has been tested on cancer cells and normal cells. Matadine is an anhydronium base as serpentine, that exerts also a selective inhibiting activity on B16 melanoma cells while it is less toxic in human 2002 non-cancer cells. This selective activity might be well due , as it seems to be the case for serpentine and alstonine, to a higher affinity of matadine for destabilized single-stranded DNA as mainly present in cancer cells.