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  • 1
    Electronic Resource
    Electronic Resource
    s.l. : American Chemical Society
    Biochemistry 20 (1981), S. 418-428 
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-0827
    Keywords: Matrix vesicles ; 32P-phosphate and45Ca-metabolism ; Epiphyseal cartilage ; Calcification ; Alkaline phosphatase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Summary Matrix vesicle-enriched fractions were isolated from different zones of epiphyseal cartilage by nonenzymatic methods involving tissue homogenization, differential centrifugation, and isosmotic Percoll gradient fractionation. Uptakes of both32Pi and45Ca were studied concomitantly over periods from 20 min to 24 h. Percoll density gradients separated epiphyseal microsomes into two alkaline phosphatase-rich fractions: a low-density noncalcifiable fraction (P-I), and a higher-density fraction (P-II) which readily mineralized. The P-II fraction was found only in calcifying regions of the growth plate. Based on chemical and physical properties and enzyme activities, both fractions were similar except that P-II contained significantly higher levels of mineral ions than did P-I, and had lower levels of alkaline phosphatase. The mineral appeared to be primarily in a noncrystalline form. Metabolism of32Pi and45Ca by P-II followed a complex kinetic pattern in which accumulation of large amounts of both ions was preceded by an initial limited burst of uptake and a lag-phase of variable duration. During mineral ion loading, the density of the P-II fraction progressively increased as evidenced by co-migration of45Ca,32Pi, and alkaline phosphatase to increasingly higher densities. During the period of early mineral deposition (1–5 h), Ca/P uptake ratios were very low (1.0–1.2) and X-ray diffraction patterns showed a predominantly amorphous pattern. This suggests that the mineral accumulated in matrix vesicles is initially some form of noncrystalline calcium monohydrogenphosphate. L-tetramisole, a potent inhibitor of alkaline phosphatase, inhibited accumulation of both45Ca and32Piin the absence of organic P substrates,32Pi being preferentially inhibited over45Ca. This finding, coupled with recent studies on the behavior of alkaline phosphatase at physiological pH, suggests that the protein is not acting as a phosphohydrolase, but rather as a Pi-binding or transport agent in vesicle-mediated calcification.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1432-0827
    Keywords: Matrix vesicles ; Acidic phospholipids ; Membrane labeling ; Mineralization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Summary The localization of serine and ethanolamine phospholipids in isolated chick epiphyseal cartilage matrix vesicles was studied by time-course labeling with trinitrobenzenesulfonate (TNBS), a probe nonpermeant to most biological membranes. Significant amounts of all four of the primary amine-containing phospholipids in matrix vesicles reacted readily with TNBS, indicating that the vesicle membranes were unusually permeable to the label. We attribute this to the presence of large amounts of lysophospholipids present in the vesicle membranes. However, none of the lipids reacted with TNBS to completion. Plateau labeling values suggested that as much as 70 to 80% of the phosphatidylserine (PS) and 60 to 80% of the lysophosphatidylserine (LPS) are located inside the vesicles, and that 40 to 50% of the ethanolamine phospholipids are internal. The kinetic evidence further suggested that an additional 10 to 15% of these phospholipids are probably also internal. In the presence of chloroform-methanol, 1:1, which totally disrupts membrane structure, only 55 to 60% of the total PS and LPS reacted with TNBS, whereas all of the ethanolamine phospholipids did. We attribute this effect to the presence of PS-Ca-Pi complexes which inhibit the reaction of these lipids with TNBS under these conditions. Thus our findings add further evidence that binding of Ca by acidic phospholipids within the matrix vesicles may be involved in mineral deposition in epiphyseal cartilage.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 26 (1978), S. 237-241 
    ISSN: 1432-0827
    Keywords: Epiphyseal chondrocytes ; Freezefracture ; Scanning electron microscopy ; Cell processes ; Membrane particles
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Summary Chondrocytes in epiphyseal cartilage were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) using freeze-fracture techniques. Freeze-fracture replicas showed large numbers of fingerlike, 0.11–0.15 μm diameter, projections from the chondrocyte surface, with numerous 95–180 Å diameter intramembranous particles associated with both the cell membrane surface and these projections. With SEM, these cytoplasmic projections were also obvious, but appeared collapsed into clusters of globular-shaped projections on the surface of the chondrocytes. With freeze-fracture techniques, in which shrinkage artifacts were essentially eliminated, the cytoplasmic projections were often seen in intimate contact with the extracapsular matrix. However, with chondrocytes prepared by both SEM and conventional TEM, there was evidence of shrinkage, the cytoplasmic projections having little contact with the extracapsular matrix. These findings show that the cytoplasmic processes are not artifacts of tissue processing and provide morphological evidence in support of the hypothesis that matrix vesicles are of cellular origin.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 4 (1969), S. 20-38 
    ISSN: 1432-0827
    Keywords: Calcification ; Epiphyseal Cartilage ; Bone ; Electrolytes ; Organic matrices
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Description / Table of Contents: Résumé Un procédé de dissection a été mis au point pour permettre l'analyse zonale du cartilage de l'épiphyse des os de la jambe d'un foetus bovin. Des échantillons de tissu complet et lavé venant des différentes zones ont été analysés pour déterminer leur contenu en électrolyte et en constituants organiques, ainsi que pour leur densité, cendres et humidité. Les résultats ont montré que lorsque la quantité de cendres et la densité augmentaient, l'eau contenu dans le tissu diminuait. Les quantités de cendres dans les zones de cartilage en voie de calcification étaient plus grandes qu'il avait été. Quand elles étaient exprimées comme un pourcentage du poids sec, elles étaient les plus importantes dans le cartilage lavé calcifié que dans le autre zones. Au début de la minéralisation du cartilage, la quantité de Na (m moles/l de tissu frais) diminuait tandis que celles du Ca et du P inorganique augmentaient. Les niveaux de Mg augmentaient pendant que la calcification se poursuivait, mais seulement à une faction du taux du Ca et du P. Les rapports Ca/P inorganique étaient les plus grands dans le cartilage au repos (Cartilage non-différentié hyalin), suggérant un lien initiale entre Ca et les chrondromucoprotéines. Cependant, au début de la calcification, pendant la prolifération du cartilage les rapports Ca/P étaient beaucoup plus petits (ca. 1.50) mais augmentaient graduellement avec l'advancement de la minéralisation. Des changements importants survenaient dans la composition de la phase organique, pendant la calcification endochondrale. Comme il a été déterminé par l'analyse de l'hydroxyproline la quantité de collagéne diminuait progressivement pendant la calcification du cartilage, mais augmentait rapidement pendant la formation d'os. Comme il a été déterminé par l'analyse de l'héxosamine et du sulfute les chrondromucoprotéines étaient aux niveaux les plus éléves pendant la prolifération du cartilage et diminuaient constamment au cours de la calcification. Cependant, bien que la calcification était déja très avancée dans le cartilage hypertrophique, de grandes quantites de mucopolysaccharides étaient encore présentes. Les rapports sulfure/hhéxosamine montraient un léger déclin pendant les premiéres étapes de la calcification, mais augmentaient beaucoup pendant le cours de la minéralisation. Les quantités d'acide sialique étaient plus grandes dans le cartilage de l'épiphyse que dans le cartilage au repos ou dans l'os. Les lipides augmentaient rapidement pendant la calcification du cartilage, mais étaient très réduites dans l'os complètement formé. La signification de ces résultats est discutée.
    Abstract: Zusammenfassung Eine Seziermethode, die eine Schichten-Analyse der Beinepiphysenplatte von Rinderfeten erlaubt, wurde entwickelt. Proben vor und nach Waschen des Gewebes der verschiedenen Schichten werden untersucht in bezug auf Elektrolyte und organische Bestandteile, als auch in bezug auf Dichte, Aschengehalt und Feuchtigkeit. Die Resultate zeigten eine Zunahme des Aschengehaltes und der Dichte, während der Wassergehalt abnahm. Unerwartet hoch waren die Aschenwerte im in Verkalkung begriffenen Knorpel. Ausgedrückt in Prozent Trockengewicht, ergab gewaschener, verkalkter Knorpel den höchsten Wert aller Zonen. In den Frühstadien der Knorpelmineralisation nahm der Natriumgehalt (m Mol/l Frischgewebe) ab, während Ca und anorganischer P zunahmen. Mit fortschreitender Verkalkung erhöhte sich auch der Magnesium-Spiegel, allerdings nur zu einem Bruchteil des Ausmaßes, in welchem Ca und P zunahmen. Die höchsten Ca/P anorg. Verhältnisse wurden im Ruheknorpel (undifferenzierter hyaliner Knorpel) gefunden, was auf eine initiale Bindung von Ca durch Chondromucoproteine hinweist. Die Ca/P-Verhältnisse proliferierenden Knorpels waren jedoch bei Verkalkungsbeginn viel tiefer (ca. 1.50). Diese nahmen allerdings mit fortschreitender Mineralisierung stetig zu. In der endochondralen Verkalkungsphase fanden markante Veränderungen in der Zusammensetzung des organischen Anteils statt. Basierend auf der Hydroxyprolinanalyse nahm der Collagengehalt in der knorpeligen Verkalkungsperiode fortschreitend ab, während er jedoch bei der Knochenbildung rasch zunahm. Die an Hand von Hexosamin- und Schwefelanalysen bestimmten Chondromucoproteingehalte ergaben Höchstwerte im proliferierenden Knorpel und fielen stetig ab mit zunehmender Verkalkung. Trotz der im hypertrophischen Knorpel schon weit fortgeschrittenen Verkalkung waren immer noch große Mengen an Mucopolysacchariden vorhanden. Die Schwefel/Hexosamin-Verhältnisse zeigten eine minimale Abnahme in den frühen Verkalkungsphasen, nahmen jedoch markant zu bei fortschreitender Mineralisation. Der Sialinsäurespiegel war im Epiphysenknorpel, verglichen mit demjenigen des Ruheknorpels oder Knochens, erhöht. In der knorpeligen Verkalkungsphase nahmen die Lipide rasch zu, während jedoch die Werte des vollständig ausgebildeten Knochens stark vermindert waren. Die Bedeutung dieser Ergebnisse wird besprochen.
    Notes: Abstract A dissection procedure has been devised to permit zonal analysis of the epiphyseal plate of fetal calf leg bones. Samples of whole and washed tissue from the various zones were analyzed for their content of electrolyte and organic constituents, as well as for density, ash and moisture. Results showed that as ash content and density increased, water content decreased. Ash levels in calcifying cartilage zones were unexpectedly high. When expressed as a percentage of dry weight, washed calcified cartilage had the highest content of any zone. In the early stages of the mineralization of cartilage, Na content (mmoles/l of fresh tissue) decreased as Ca and inorganic P increased. Magnesium levels increased as calcification proceeded, but only at a fraction of the rate of Ca and P. Ratios of Ca/inorganic P were highest in resting cartilage (non-differentiated hyaline cartilage), suggesting an initial binding of Ca to chondromucoproteins. However, at the onset of calcification in proliferating cartilage, Ca/P ratios were much lower (ca. 1.50), but gradually increased with advancing mineralization. Marked changes occurred in the composition of the organic phase during endochondral calcification. As determined by hydroxyproline analysis, collagen content progressively decreased during cartilaginous calcification, but increased rapidly during bone formation. As determined by hexosamine and sulfur analysis, chondromucoproteins were at highest levels in proliferating cartilage and decreased steadily as calcification increased. However, although calcification was already well advanced in hypertrophic cartilage, large amounts of mucopolysaccharide still were present. Sulfur/hexosamine ratios showed a slight decline during the early stages of calcification, but increased markedly with further mineralization. Sialic acid levels were elevated in epiphyseal cartilage over those in resting cartilage or bone. Lipids increased rapidly during cartilaginous calcification, but were greatly reduced in fully-formed bone. The significance of these findings is discussed.
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  • 6
    ISSN: 0886-1544
    Keywords: chondrocytes ; matrix vesicle formation ; actin ; tubulin ; myosin ; vinculin ; alkaline phosphatase ; immunofluorescence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Matrix vesicles, extracellular microstructures known to eb involved in endochondral calcification, are rich in alkaline phosphatase and have been shown to contain actin. The mechanism of matrix vesicle formation in chondrocytes in not well understood. Chondrocytes from the epiphyseal growth plate, when grown in primary culture, elaborate alkaline phosphatase-rich vesciles. We examined the distribution of the cytoskeletal proteins actin, myosin, tubulin, and vinculin at various time-points during culture using indirect immunofluorescent labeling. Concomitantly, the production of alkaline phosphatase-containing matrix vesicles was also followed. Cell morphology changed noticeably at two distinct stages during the 22-day culture period: Immediately after release from the growth plate the cells were founded, but after 4 days of cultre they began to spread out and acquire irregular shapes with distinct filopodia. By 13 datsm as tge cekks attaubed confluency, they reacquired a rounded, polygonal appearance. At all time-point, tubulin was seen as a dense network of microtubules radiating from the perinuclear region throughout the cytoplasm toward the cell periphery. Initially actin was seen in filamentous from, but displayed a punctate distribution focused at contact points during the cell-spreading stage of culture. After confluency, actin was concentrated at cell-cell junctions. Initially, vinculin was diffusely distributed, but became focused in multiple adhesion plaques and at the termini of filpodia during the cell-spreading stage of culture. Following confluency vinculin became concentrated at cell-cell junctions. Myosin was observed at all time-points in small, intensely localized focal points in the cytoplasmic region of the cells and was consistently absent from the nuclear and peripheral regions. The amount of myosin in the cells increased steadily with time in culture. Elaboration of alkaline phosphatase-rich vesicles, which corresponded closely with the rounded morphology of early and late stages of culture, may be correlated with contact inhibition.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    The @Anatomical Record 199 (1981), S. 449-457 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The morphological features of avian epiphyseal cartilage have been investigated by freeze-fracture techniques. Progressive changes occurred in both the cells and the matrix during differentiation. Chondrocytes changed in shape from small flattened cells with few, short cellular processes, to enlarged ovoid cells with numerous long processes often associated with extracellular vesicles. In the matrix these vesicles appeared first in the cellular lacunae, then in the extralacunar matrix, becoming larger and more numerous. Large membrane-associated particles (MAPS) were seen on the p faces of the plasmalemma. These became progressively concentrated on and around the cellular processes, with few large MAPS being seen on the e face. Similar distribution of MAPS was seen in the matrix vesicles. Domains of hydrated proteoglycan aggregates were manifest as regular fracture patterns in the extralacunar matrix of the upper regions of the plate. Collagen fibrils progressively increased in size and state of aggregation, often being associated with matrix vesicles and in the end, with long plate-like mineral crystals. These findings, while in basic agreement with patterns observed with TEM, reveal important new features concerning cellular and matrix structure during cartilage differentiation.
    Additional Material: 11 Ill.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 49 (1992), S. 181-198 
    ISSN: 0730-2312
    Keywords: chondrocytes ; TGF-β1 ; bFGF ; collagen ; fibronectin ; alkaline phosphatase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Expression of several cellular and matrix proteins which increase significantly during the maturation of growth plate cartilage has been shown to be affected by various endocrine and autocrine factors. In the studies reported here, transforming growth factor-β (TGF-β1) and basic fibroblast growth factor (bFGF) were administered to primary cultures of avian growth plate chondrocytes at pre- or post-confluent stages to study the interplay that occurs between these factors in modulating chondrocytic phenotype. Added continuously to pre-confluent chondrocytes, TGF-β1 stimulated the cells to produce abundant extracellular matrix and multilayered cell growth; cell morphology was altered to a more spherical configuration. These effects were generally mimicked by bFGF, but cell shape was not affected. Administered together with TGF-β1, bFGF caused additive stimulation of protein synthesis, and alkaline phosphatase (AP) activity was markedly, but transiently enhanced. During this pre-confluent stage, TGF-β1 also increased fibronectin secretion into the culture medium. Added to post-confluent cells, TGF-β1 alone caused a dosage-dependent suppression of AP activity, but bFGF alone did not. Under these conditions, TGF-β1 and bFGF had little effect on general protein synthesis, but TGF-β1 alone caused large, dosage-dependent increases in synthesis of fibronectin, and to some extent type II and X collagens. Given together with bFGF, TGF-β1 synergistically increased secretion of fibronectin. These findings reveal that regulation of phenotypic expression in maturing growth plate chondrocytes involves complex interactions between growth factors that are determined by timing, level, continuity, and length of exposure.
    Additional Material: 12 Ill.
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  • 9
    ISSN: 0730-2312
    Keywords: chondrocytes ; cell culture ; mineralization ; calcospherites ; Ca and P mapping ; matrix vesicles ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Advances in the culture of mineralizing growth plate chondrocytes provided an opportunity to study endochondral calcification under controlled conditions. Here we report that these cultures synthesize large amounts of proteins characteristically associated with mineralization: type II and X collagens, sulfated proteoglycans, alkaline phosphatase, and the bone-related proteins, osteonectin and osteopontin. Certain chondrocytes appeared to accumulate large amounts of Ca2+ and Pi during the mineralization process: laser confocal imaging revealed high levels of intracellular Ca2+ in their periphery and X-ray microanalytical mapping revealed the presence of many Ca2+- and Pi-rich cell surface structures ranging from filamentous processes 0.14 ± 0.02 μm by 0.5-2.0 μm, to spherical globules 0.70 ± 0.27 μm in diameter. Removal of organic matter with alkaline sodium hypochlorite revealed numerous deposits of globular (0.77 ± 0.19 μm) mineral (calcospherites) in the lacunae around these cells. The size and spatial distribution of these mineral deposits closely corresponded to the Ca2+-rich cell surface blebs. The globular mineral progressively transformed into clusters of crystallites. Taken with earlier studies, these findings indicate that cellular uptake of Ca2+ and Pi leads to formation of complexes of amorphous calcium phosphate, membrane lipids, and proteins that are released as cell surface blebs analogous to matrix vesicles. These structures initiate development of crystalline mineral. Thus, the current findings support the concept that the peripheral intracellular accumulation of Ca2+ and Pi is directly involved in endochondral calcification.
    Additional Material: 13 Ill.
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  • 10
    ISSN: 0730-2312
    Keywords: retinoic acid ; chondrocytes ; weight-bearing joints ; proteoglycan synthesis ; proteoglycan depletion ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The effect of retinoic acid (RA) on primary cultures of growth plate chondrocytes obtained from weight-bearing joints was examined. Chondrocytes were isolated from the tibial epiphysis of 6- to 8-week-old broiler-strain chickens and cultured in either serum-containing or serum-free media. RA was administered at low levels either transiently or continuously after the cells had become established in culture. Effects of RA on cellular protein levels, alkaline phosphatase (AP) activity, synthesis of proteoglycan (PG), matrix calcification, cellular morphology, synthesis of tissue-specific types of collagen, and level of matrix metalloproteinase (MMP) activity were explored. RA treatment generally increased AP activity, and stimulated mineral deposition, especially if present continuously. RA also caused a shift in cell morphology from spherical/polygonal to spindle-like. This occurred in conjunction with a change in the type of collagen synthesized: type X and II collagens were decreased, while synthesis of type I collagen was increased. There was also a marked increase in the activity of MMP. Contrasting effects of continuous RA treatment on cellular protein levels were seen: they were enhanced in serum-containing media, but decreased in serum-free HL-1 media. Levels of RA as low as 10 nM significantly inhibited PG synthesis and caused depletion in the levels of PG in the medium and cell-matrix layer. Thus, in these appendicular chondrocytes, RA suppressed chondrocytic (PG, cartilage-specific collagens) and enhanced osteoblastic phenotype (cell morphology, type I collagen, alkaline phosphatase, and mineralization). J. Cell. Biochem. 65:209-230. © 1997 Wiley-Liss, Inc.
    Additional Material: 11 Ill.
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