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PubMed1979

Stimulation of chondroitin sulfate synthesis by beta-D-xyloside in chondrocytes of the proteoglycan deficient mutant nanomelia.

The potential of nanomelic chondrocytes to synthesize chondroitin sulfate was investigated by providing the mutant cells with p-nitrophenyl-beta-D-xyloside, a compound which acts as an artificial acceptor for glycosaminoglycan synthesis. Under these conditions the synthesis of chondroitin sulfate in nanomelic and normal chondrocytes is comparable. The chondroitin sulfate synthesized by the mutant is indistinguishable in molecular size and composition from that synthesized by similarly treated normal chondrocytes.

Animals↗

PubMed1979

Long acting cAMP analogues enhance sulfate incorporation into matrix proteoglycans and suppress cell division of fetal rat chondrocytes in monolayer culture.

The relationship between replication and the synthesis of matrix sulfated proteoglycans was investigated with fetal rat chondrocytes grown in monolayer culture. The effect of N6 O2' dibutyryl adenosine 3', 5' cyclic monophosphate (DBcAMP), adenosine 3', 5' cyclic monophosphate (cAMP), 8 Bromo adenosine 3', 5' cyclic monophosphate (8 Br-cAMP), sodium butyrate and hydroxyurea was examined. Between 0.05 and 0.5 mM DBcAMP, a dose related inhibition of cell division and stimulation of [35SO=/4] incorporation into matrix proteoglycans was demonstrated. At the higher concentrations of DBcAMP, cell division was completely inhibited and the enhancement of [35SO=/4] incorporation into matrix proteoglycans ranged between 40 and 120% (P less than 0.01). Utilizing 14C-glucosamine and photometric determination of proteoglycans with Alcian Blue, it was demonstrated that the increase in sulfate incorporation reflected enhanced accumulation of extracellular matrix. The effects of DBcAMP were mimicked by 8 Br-cAMP, suggesting they were mediated by the adenylyl cyclase system. cAMP (0.05-0.5 mM), sodium butyrate (0.1-0.5 mM) and hydroxyurea (0.5-5 mM) partially or fully inhibited cell division, but either failed or only slightly enhanced sulfate incorporation. The enhanced sulfated proteoglycan deposition promoted by DBcAMP began 8 to 12 hours after serum stimulation, its onset occurred prior to thymidine incorporation and the effect persisted for 28 hours. Determination of cell volume demonstrated an increase in size of DBcAMP treated chondrocytes between 8 to 12 hours, coincident with the onset of increased sulfate incorporation. These results are consistent with a model where matrix sulfated proteoglycan deposition by chondrocytes is mediated by intracellular cAMP levels and occurs in the G1 phase of the cell cycle.

Animals↗

PubMed1978

Ca2+ transport by chondrocyte mitochondria of the epiphyseal growth plate.

In a study of the Ca2+ kinetics of mitochondria of chick epiphyseal chondrocytes, the rate of Ca2+ uptake was linear up to a medium Ca2+ concentration of 30 mum. The half maximal transport rate occurred at 34 mum Ca2+. The Ca2+ uptake rate, expressed as a function of time, was 35 nmoles/mg protein/min; the presence of Mg2+ had little effect on Ca2+ accumulation. While these kinetic parameters did not differ significantly from mitochondria of cells of nonmineralizing tissues, the respiratory characteristics of the chondrocyte organelles exhibited functional differences. Thus, up to 350 nmoles Ca2+/mg protein, chondrocyte mitochondria performed coupled oxidative phosphorylation. Calcium uptake was energy supported, while Ca2+ binding was low. Addition of respiratory inhibitors and uncouplers to these mitochondria resulted in a rapid loss of more than 80% of the total Ca2+. The Ca/Pi ratio of the extrudate was very similar to the ratio of the ions in cartilage septum fluid. In the most mineralized zones of the epiphyseal plate, there was little change in the state 4 respiratory rate, but nonspecific Ca2+ binding was elevated and a high percentage of the total Ca2+ was in a nonextrudable form. The results indicate that in cells preparing for mineralization, much of the total mitochondrial Ca2+ is in a form that can be transported to the calcification front. In cells close to the calcification front, nonextrudable Ca2+ may form calcium phosphate granules described by other investigators.

Animals↗

PubMed1977

Species differences in cell culture of mammalian articular chondrocytes.

Articular chondrocytes from eight mammalian species (rabbit, opossum, woodchuck, cat, dog, sheep, rhesus and cebus monkeys) were grown in monolayer culture using a single regimen. The animals were immature or young adults. Ham's F12 medium supplemented with 10% fetal bovine serum was employed for the primary cultures and Dulbecco-Vogt medium, for the secondary. Marked species differences were found with respect to cell morphology, growth in primary and secondary cultures, incorporation of radiosulfate into macromolecules, adhesion to the flask surface, response to vitamin C, and chondroid expression in spinner bottles. Under these particular conditions, rabbit chondrocytes grew most rapidly and incorporated several times more sulfate than did the others. Additional experiments carried out with other media on four of the species indicate that optimal conditions for culturing mammalian chondrocytes must be determined for each species individually.

Animals↗

PubMed1977

The proliferation of chondrocytes and pannus in adjuvant arthritis.

Cell proliferation in the pannus formation of adjuvant arthritis was studied by autoradiography. It was found that after day 9 an increased cell proliferation starts in the joint capsule recessus and synovial villi on the injected side. From these proliferating cells a pannus, which during the first phase frequently consists only of few cell layers, extends over the cartilage surface. With advancing disease the thickness of the pannus increases and further centripetal growth may cause the entire cartilage surface to be covered. This proliferating pannus tissue may invade the cartilage and destroy it. Since in this area of destruction labelled cells are frequently present, it may be assumed that proliferating cells with a high enzyme content are particularly responsible for the immediate degradation of cartilage. No involvement of chondrocytes in pannus formation was confirmed by the methods employed. There was neither increased proliferation of surface chondrocytes nor increased proliferation of chondrocytes in the depth of cartilage.

Animals↗

PubMed1979

Enzymatic characterization of the chondrocytic alkaline phosphatase isolated from bovine fetal epiphyseal cartilage.

Purified chondrocytic alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and inorganic pyrophosphate. Optimal activities for p-nitrophenyl phosphate, ATP and inorganic pyrophosphate are found at pH 10.5, 10.0 and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations. p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasng substrate concentration. Heat inactivation studies indicate that both phosphorolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg2+ (0.1-10.0 mM) and Mn2+ (0.01-0.1 mM) show a small stimulation of p-nitrophenyl phosphate-splitting activity at pH 10.5. Levamisole, Pi, CN-, Zn2+ and L-phenylalanine are all reversible inhibitors of the phosphomonoesterase activity. Pi is a competitive inhibitor with a Ki of 10.0 mM. Levamisole and Zn2+ are potent non-competitive inhibitors with inhibition constants of 0.05 and 0.04 mM, respectively. The chondrocytic alkaline phosphatase is inhibited irreversibly by Be2+, EDTA, EGTA, ethane-1-hydroxydiphosphonate, dichloromethane diphosphonate, L-cysteine, phenyl-methylsulfonyl fluoride, N-ethylmaleimide and iodoacetamide. NaCL, KCL and Na2SO4 at 0.5-1.0 M inhibit the enzyme. At pH 8.5, the cleavage of inorganic pyrophosphate (pyrophosphate phosphohydrolase, EC 3.6.1.1) by the chondrocytic enzyme is slightly enhanced by low levels of Mg2+ and depressed by concentrations higher than 1mM. Ca2+ show only inhibition. Similar effects of Mg2+ and Ca2+ on the associated ATPase (ATP phosphohydrolase, EC 3.1.6.3) activity were observed. Arrhenius studies using p-nitrophenyl phosphate and AMP as substrates have accounted for the ten-fold difference in V in terms of small differences in both the enthalpies and entropies of activation which are 700 cal/mol and 2.3 cal/degree per mol, respectively.

Adenosine Triphosphatases↗

PubMed1979

Simultaneous localization of type II collagen and core protein of chondroitin sulfate proteoglycan in individual chondrocytes.

In order to investigate the coordinated synthesis of matrix components by individual chondrocytes, specific antibodies to type I collagen, type II collagen, and chondroitin sulfate proteoglycan core protein were used in simultaneous double immunofluorescence reactions. Extensive accumulation of core protein surrounding chondrocytes and the intracellular accumulation of type II collagen were observed. Extracellular core protein immunofluorescence obscured the intracellular reaction product, but the extracellular immunoreactive material could be removed by digestion with purified testicular hyaluronidase prior to fixation. Subsequent to digestion, core protein and type II collagen were observed in the same chondrocytes within discrete, sometimes identical, cytoplasmic regions, thus demonstrating the simultaneous localization of these two products characteristic of differentiating cartilage.

Animals↗

PubMed1979

Effects of colchicine on endocytosis and cellular inactivation of horseradish peroxidase in cultured chondrocytes.

Horseradish peroxidase (HRP) was used as a marker to study the effects of microtubule-disruptive drugs on uptake and cellular inactivation of exogenous material in cultures of embryonic chick chondrocytes. HRP was ingested by fluid endocytosis, and intracellular enzyme activity subsequently diminished exponentially with time. Cytochemically, reaction product for HRP was found in vesicles often located close to the dictyosomes of the Golgi complex. Colchicine and vinblastine caused disappearance of cytoplasmic microtubules and disorganization of the Golgi complex with concomitant reduction in the cellular uptake of HRP to about half of that in the controls. Lumicolchicine, on the other hand, left cell fine structure and HRP uptake unaffected. These results indicate that microtubules are of considerable importance in the process of fluid endocytosis in cultured chondrocytes although the exact mechanism remains to be elucidated. The rate of intracellular inactivation of ingested HRP was not affected by colchicine or vinblastine. Double-labeling experiments with colloidal thorium dioxide and HRP likewise indicated that fusion of endocytic vesicles and lysosomes is not dependent on intact microtubules. The total specific activities of the three lysosomal enzymes examined were weakly or not at all changed by treatment of the cultures with colchicine or vinblastine. It therefore seems unlikely that microtubular organization plays an important role in the production or degradation of lysosomal enzymes in cultured chondrocytes.

Acetylglucosaminidase↗

PubMed1977

Transformation and neoplastic development of hamster chondrocytes after exposure to 4-nitroquinoline-1-oxide and 3-methylcholanthrene in tissue culture.

Sternal hyaline cartilages of Syrian hamsters were dissociated with collagenase and culured. Primary monolayer cultures of the dissociated cells were morphologically homogeneous. Secondary cultures of the chondrocytes were treated with 1 x 10(-6) or 2 x 10(-6) M 4-nitroquinoline-1-oxide (4NQO) for 3 hours or with 5 or 10 microgram 3-methylcholanthrene (MCA)/ml for 3 days. Cultured chondrocytes transformed morphologically 29-51 days after 4NQO treatment and 41-61 days after MCA treatment and began to grow continuously in vitro. Cultures of transformed cells, like transformed fibroblasts. Untreated cells and cells treated with dimethyl sulfoxide did not transform within at least 110 days after inoculation. Among the transformed cells, one near-diploid cell line preserved the distinct phenotypic expression of chondrocytes, whereas heteroploid cell lines lost their differentiated features. The near-diploid cell line produced nodules in the cheek pouches of hamsters within a week, by the nodules regressed later. They showed the chondrogenic properties of the original cells and stained metachromatically with toluidine blue. Heteroploid cell lines formed progressive tumors with few chondrogenic features; these tumors were diagnosed as fibrosarcomas.

4-Nitroquinoline-1-oxide↗

PubMedDate not supplied

Chondrocyte-to-osteocyte transformation in grafts of perichondrium-free epiphyseal cartilage.

When perichondrium-free pieces of embryonic quail epiphyseal cartilage are incubated on the chorioallantoic membranes of chick embryos, 2 developmental changes are observed. First, most grafts develop a periosteum in which the osteoblasts and osteocytes are of donor, i.e., chondrocytic origin. No such periosteum is observed around explants of demineralized, inductive bone matrix. Second, the matrix surrounding some chondrocytes within the original graft became more bone-like with respect to staining pattern, birefringence and collagen morphology. We conclude that, under some conditions, the avian chondrocyte may in situ or subsequent to release from the cartilage lacuna synthesize a bone-like matrix and, in this sense, be thought to have undergone a "transformation" into an osteocytic or osteoblastic type of cell.

Allantois↗

PubMed1976

Light microscopic localization of labile calcium in hypertrophied chondrocytes of long bone with alizarin red S.

A method which localizes labile 5% ethylene glycol-bis-(beta-amino-ethyl ether)N-N'-tetraacetic acid-removable calcium in spherules within hypertrophied chondrocytes and in pericellular matrix using alizarin red S (ARS) is described. Fresh blocks of epiphyseal cartilage approximately 1 mm thick were immersed into 0.5-2% ARS solution containing 7% mounted on glass slides in 7% sucrose or in glycerol-gelatin. The stained tissue blocks were also dehydrated in acetone, cleared in xylene and mounted in Preservaslide. The ARS precipitated ionic calcium as red Ca-ARS salt which was birefringent in polarizing microscope, stable in water at pH 4-9 and in nonpolar organic solvent but soluble in polar solvents, especially in dimethyl sulfoxide. In contrast, ARS-stained insoluble calcium phosphate was stable even in dimethyl sulfoxide. Calcium in the hypertrophied chondrocytes, therefore, was thought to be present in a readily ionizable state instead of as insoluble calcium phosphate. Since addition of 7% sucrose retained as well as improved ARS localization of cellular calcium, the calcium was believed to be present in an osmotically sensitive, membrane-bound cytoplasmic compartment. The ARS-positive labile calcium in spherules which develop in the hypertrophied chondrocytes as well as in the pericellular matrix at the zone of provisional calcification suggested a preparatory stage in the process of cartilage calcification.

Animals↗

PubMed1975

Pituitary fibroblast growth factor as a stimulator of growth in cultured rabbit articular chondrocytes.

Growth promoting activity for rabbit chondrocytes has been described as a contaminant of partially pruified TSH and LH prepared from bovine and ovine pituitaries. We have investigated fibroblast frowth factor (FGF), a small growth promoting peptide isolated from bovine pituitary tissue, in a rabbit chondrocyte system. The results suggest to us that FGF is the factor or one of the factors responsible for chondrocyte growth stimulating activity previously described in the pituitary hormone preparations. DNA synthesis in these cells is stimulated by FGF at final medium concentrations of 10(-9) g/ml. Bovine NIH-LH is not stimulatory below concentrations of 10(-7) g/ml. FGF also stimulates cell growth in the presence of 10% fetal bovine serum. Dexamethasone, at concentrations of 10(-5) to 10(-9) g/ml exerts a synergistic effect with FGF on both DNA synthesis and cell growth. Over a concentration range of 10(-6) to 10(-9) g/ml, FGF does not stimulate synthesis of sulfated mucopolysaccharides.

Animals↗

PubMed1977

Synthesis of proteoglycans by suspension and monolayer cultures of adult chondrocytes and de novo cartilage nodules-the effect of hyaluronic acid.

Chondrocytes were isolated from adult laryngeal cartilage by an enzymic procedure that included 6 h digestion with collagenase. The level of 35SO4(2-) incorporation into cetylpyridinium chloride-precipitable material by these cells depended upon the subsequent culturing conditions. Suspension cultures incorporated more 35SO4(2-)/cell than monolayer cultures. Hyaluronic acid in the medium inhibited 35SO4(2-) incorporation only when the cells were in primary suspension cultures. It had no effect on monolayer cultures, or monolayers organized into nodules, or suspension cultures derived from monolayers. Mild pretreatment with EDTA, however, rendered these susceptible to hyaluronic acid inhibition. In contrast EDTA abolished the inhibitory effect of hyaluronic acid on primary suspension cultures. Oligosaccharides, derived from hyaluronidase digestion of hyaluronic acid that were larger than decassaccharide, had some inhibitor effect on 35SO4(2-) incorporation by monolayer cultures. The total 35SO4(2-) incorporation was less in primary suspension cultures of chondrocytes isolated after 12 h than after 6 h digestion of cartilage and the inhibition by hyaluronic acid was also less. These differences persisted during 12 days of culture. It is suggested that the method of isolating chondrocytes and subsequent culture conditions may modify the cell surface and mask or abolish specific binding sites for hyaluronic acid.

Animals↗

PubMed1978

The effect of a slightly acidic somatomedin peptide (ILAs) on the sulphation of proteoglycans from articular and growth plate chondrocytes in culture.

Chondrocyte cultures were prepared from rabbit growth plate (GPC) and articular (ARC) chondrocytes. These two cell types have distinct morphological characteristics. The cells reached maximum numbers by days 10 and 21 for ARC and GPC, respectively. The proteoglycans (PG) contained in the cellular pool were extracted and purified by DEAE cellulose chromatography. The effect of a partially purified somatomedin peptide with insulin-like activity on [35S]sulphate incorporation into PG was evaluated. In both ARC and GPC a significant stimulation of [35S]sulphate uptake into PG subunits was obtained with 1 ng Eq./ml of somatomedin peptide. In order to obtain the same stimulatory effect with porcine insulin, a 1000-fold greater concentration was required. The electrophoretic patterns of the PG subunits on acrylamide-agarose electrophoresis were identical on control incubations and after stimulation with the somatomedin peptide. These data demonstrate in vitro biological activity of this peptide on well differentiated articular and epiphyseal growth plate chondrocytes in culture. These cultures appear to provide a sensitive biological assay for somatomedin peptides.

Animals↗

PubMed1977

UDP-glucuronate carboxy-lyase in cultured chondrocytes.

UDP-glucuronate carboxy-lyase has been demonstrated in chick chondrocytes in tissue culture. It occurs in the particulate fraction, and its activity is stimulated by exogenous NAD. The enzyme is allosterically activated by UDP-glucuronate and inhibited by UDP-xylose, n Values of 2.8 indicate positive cooperativity of at least three interacting sites on the enzyme. These data suggest that UDP-xylose concentration in chondrocytes is regulated by substrate activation and product inhibition of UDP-glucuronate carboxy-lyase. Activity levels of the enzyme during growth of the cells peak towards mid-log phase and decline thereafter, closely paralleling levels of chondroitin sulfate glycosyltransferases determined previously (Schwartz, N. B. (1976) J. Biol. Chem. 251, 3346-3351). Thus, it appears that during chondrocyte development a common mechanism governs induction of glycosyltransferases and of UDP-glucuronate carboxy-lyase.

Animals↗

PubMed1977

[The fixation of intracellular carbohydrates in the chondrocytes (author's transl)].

The chondrocytes of the epiphysis contain several polysaccharides, not only glycogen, but also proteoglycans (MPS). Therefore the fixation of chondrocytes is difficult. In regard of fixation we must consider, that macromolecules of proteoglycans have a considerable water content (the effective hydrodynamic volume), furthermore the MPS are easily soluble. We examined the preservation of chondrocytes after application of 44 several fixation fluids with the aid of phasecontrast, the electronmicroscope and after staining. The fixation with familiar methods is unsufficient. Consequently we used a double or successive fixation with two different mixtures, once for fixation the proteins (formalin-alcohol-mercuric chloride-acetic acid), another for polysaccharides (LILLIE's alcoholic lead nitrate formalin alternative basic lead acetate). The degree of fixation, the precipitability of the various polysaccharides is quite different and may be characteristic like their staining or solubility feature.

Animals↗

PubMed1979

[In vitro study of isolated chondrocytes concerning the prognosis of transplanted cartilage].

In order to find out reliable criteria to conclude on the prognosis of transplanted cartilage the quality of isolated rabbit chondrocytes was assayed in vitro. Using a semisolid medium a culture system is introduced, which is able to detect colony forming units of chondrocytes. Furthermore their proliferative and differentiating capacity was investigated. Colony formation in vitro follows a strict dose response relationship. An inverse relationship was observed between the age of the donor and the colony incidence in vitro. Cells from neonatal cartilage could be induced to grow as fibroblastic colonies in the presence of conditioned medium from fibroblastic monolayer cultures. No effect, however, could be observed with chondrocyte derived conditioned medium.

Age Factors↗

PubMed1978

The differentiation and calcification of chondrocytes in primary cell cultures.

The cartilage from a non-immobilized fracture undergoes a series of morphological and biochemical changes resembling the in vivo differentiation and calcification in the epiphyseal plate. The studies reported here demonstrate that a homogeneous population of chondrocytes isolated from fracture callus fibrocartilage undergoes the same changes in vitro. Chondrocyte primary cultures were grown for 28 days during which time the morphological, histological and histochemical properties of the cultures were studied. Demonstrated by various histological procedures, chondrocytes synthesized the characteristic cartilage matrix, and progressively calcified with increased culture age. This system can be used to elucidate the cellular and molecular mechanisms of calcification.

Animals↗