Composition of epiphyseal cartilage

Parathyroidectomy of immature chicks and subsequent administration of parathormone effected, within 24 hours, changes in the composition of the epiphyseal cartilage. These changes can be summarized as follows:

1. Parathyroidectomy increased the levels of collagen constituents and decreased those of calcium, while the levels of hexosamines and of ash remained the same as in controls.
2. Parathormone corrected the effect of parathyroidectomy on the collagen constituents; the levels of calcium and of ash were increased above those of the intact chicks; the levels of hexosamines remained unchanged.

     

Lysozyme in epiphyseal cartilage

Short-term incubation of organ cultures of puppy scapulae in a medium enriched with lysozyme or protamine induces specific alterations in the histologic staining properties of the explant not induced by albumin or by ultraviolet-inactivated lysozyme. The induced changes are different for each two proteins. The lysozyme effect is anatomically distributed precisely while that of protamine is diffuse. If fluorescein conjugates of the proteins are used, identical histologic changes are induced in the cartilage and the distribution of fluorescence is precisely that of the altered tinctorial properties of the tissue. The observation, which shows that lysozyme, when added to cartilage, accumulates in specific anatomic sites suggests that it chemically interacts with specific components of cartilage matrix, presumably with negatively charged macromolecules.

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Zusammenfassung lysozym und Protamin angereicherte Gewebekulturen des wachsenden Hundeschulterblattes erzeugen nach kurzer Zeit spezifische Veränderungen in der histologischen Anfärbbarkeit des Explantats, die jedoch nicht durch Albumin oder durch Lysozym, das durch Ultraviolettbestrahlung inaktiviert wurde, induziert werden können. Die hervorgerufenen Veränderungen sind für beide Proteine verschieden. Der Lysozymeffekt ist anatomisch genau verteilt, während der Protamineffekt zerstreut erscheint. Fluoresceinkonjugate dieser Proteine erzeugen identische Veränderungen im Knorpel, wobei die Verteilung der Fluorescenz mit der der Veränderungen der histologischen Anfärbbarkeit übereinstimmt. Die Beobachtungen zeigen, daß durch die Zugabe von Lysozym zu Gewebekulturen dieses Protein an speziellen anatomischen Stellen angereichert wird. Es kann daraus geschlossen werden, daß Lysozym chemisch sich mit spezifischen Komponenten des Knorpels verbindet, wahrscheinlich den negativ geladenen Makromolekülen.

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Résumé Court term d'incubation des cultures d'un organe du cartilage scapulaire chez le jeune chien dans un milieu raffiné avec la lysozyme ou la protamine produit des histologiques changements spécifiques dans les proprietés de la coloration du l'explant qui n'est pas produit par l'albumine ou par la lysozyme inactivée par les rayons ultraviolets. Ces modifications sont différent avec chacune des protéines. L'effet de la lysozyme, est précisement distributé tandis que la protamine est diffusée. Si on use la flouresceine avec les protéins, les même changements histologiques se produisent dans le cartilage et la flourescence c'est repandue précisement au même endroit, où les propriétés colorant du tissue ont été changé.L'observation qui montre que la lysozyme dans des spécifiques points anatomiques suggère qu'elle réagit chimiquement avec les constituants spécifiques de la matrice cartilagineuse, probablement avec des macromolécules négativement chargées.

     

Patterns of proteoglycan degradation by a neutral protease from human growth-plate epiphyseal cartilage

The hypothesis is widely held that proteolytic degradation of proteoglycans in the lower hypertrophic zone of the growth plate may be involved in the initiation of mineralization in the zone of provisional calcification. However, a neutral protease that is responsible for the degradation of proteoglycans in the growth plate has not been identified, isolated, and characterized. In the work reported here, neutral protease activity in the growth plate is demonstrated for the first time, and some of the properties of the enzyme are described. Proteoglycans subunits were prepared from bovine nasal cartilage and calf costal cartilage by equilibrium density-gradient centrifugation under dissociative conditions. The proteoglycan subunits were labeled with 14C-formaldehyde. Homogenates from human growth plates were examined for neutral protease activity using the proteoglycan subunits as substrates. Following incubation of the proteoglycan subunits with growth-plate homogenates at pH 5.3 and at pH 7.5 in the presence and absence of ten-millimolar magnesium chloride and calcium chloride, the digestion products were examined by gel chromatography on Sepharose-2B and 6B columns. Column eluants containing proteoglycan-subunit degradation products were monitored for uronic acid, hexose, and radio-activity. Maximum extensive degradation of proteoglycan subunits occurred at pH 7.5 in the presence of ten-millimolar magnesium chloride and calcium chloride.     

Human cartilage stiffness. In vivo studies

Integrated structural properties of cartilage, the combined structural behavior of a material (which accounts for both of its elastic properties and geometry) were measured in 25 autopsies and 35 open operations. The intraoperative measurements were made at the time of total knee arthroplasties, synovectomies, amputations, and traumatic lesions of the knee. The results obtained through evaluation of force deformation curves have been computer analyzed. There were no statistically significant differences between the stiffness of autopsy cartilage and in vivo cartilage as obtained through measurements at surgery. There was significant age dependence of stiffness of joint cartilage. Stiffness of intraoperative as of autopsy cartilage, depends on the thickness of the material tested.     

Fractures of the proximal tibial epiphyseal cartilage]

The authors present 9 children who sustained 10 fractures of proximal tibial epiphyseal cartilage (7--left tibia, 3--right tibial), and were treated in Children's University Hospital in Kraków between years 1994-2001. Mean age in the time of trauma was 12.7 years (6-17 years), the mean follow-up time was 17.4 months (8-48 months). According to Salter-Harris classification there was 5 fractures of type I, 4 of type II, 1 of type III. One child was treated by closed reduction and percutaneous Kirschner wires fixation. One child was treated by traction. The rest of the children were treated by casting, in knee flexion about 30 degrees. In one child there was premature closing of epiphyseal cartilage, shortening 1.5 cm and posterior curvature of tibia. In this child we performed flexion tibial osteotomy. In one child few months later we recognized meniscal tears (the patient was operated on). The results in the rest of children were good and there were not complications. The authors discuss the literature connecting to proximal tibial epiphyseal fractures.     

Ultrastructural effects of testosterone on epiphyseal cartilage

Although experimental and clinical experience indicates that large doses of testosterone lead to premature cessation of growth, the exact mechanism and precise site of action of this hormone on the growth apparatus of long bones remain unknown. In this study, plateaued male rats were injected with supraphysiologic doses of testosterone to observe the submicroscopic effects on the various zones of the epiphyseal cartilage. In the zone of cell division there were increased numbers of dividing cells. The maturing cells accumulated larger amounts of secretory products at earlier stages of their life cycle, and appeared to undergo a more abrupt hypertrophy. In the zone of prehypertrophy, the interterritorial matrix contained foci of early and premature calcification and thicker and longer collagen fibers than at comparable levels in controls.It is concluded that in intact animals, even large doses of testosterone initially cause a stimulation of chondrocyte proliferation, prior to promoting maturation processes.

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Zusammenfassung Obwohl experimentelle und klinische Erfahrung darauf hinweisen, daß hohe Dosen von Testosteron zu einem frühzeitigen Wachstumsabschluß führen, sind der genaue Mechanismus und der eigentliche Wirkungsort dieses Hormons im Wachstumsapparat der Röhrenknochen unbekannt geblieben. In diesem Experiment wurden 200 g schweren männlichen Ratten supraphysiologische Testosterondosen injiziert, um die submikroskopischen Auswirkungen auf die verschiedenen Zonen des Epiphysenknorpels zu beobachten. In der Zone der Zellmitosen fand sich eine erhöhte Anzahl von sich teilenden Zellen. Die reifenden Zellen häuften im Frühstadium ihres Lebenscyclus größere Mengen von Sekretionsprodukten an und schienen eine abruptere Hypertrophie durchzumachen. In der prähypertrophen Zone enthielt die interterritoriale Matrix Herde von früher und verfrühter Verkalkung, sowie dickere und längere Kollagenfasern als vergleichsweise in Kontrolltieren.Daraus wird geschlossen, daß bei unbehandelten Tieren sogar große Testosterondosen anfänglich eine Stimulation der Chondrocytenproliferation verursachen, bevor sie die Reifungsprozesse veranlassen.

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Résumé Bien que la clinique et l'expérimentation semblent démontrer que des doses élevées de testostérone provoquent un arrêt prématuré de la croissance, le mécanisme exact et le lieu précis de son action sur l'appareil de croissance des os longs restent indéterminés. Au cours de cette étude, des rats máles de 200 g sont injectés à l'aide de doses supra-physiologiques de testostérone pour observer les effects sub-microscopiques sur les diverses zones du cartilage épiphysaire. Au niveau de la zone de division cellulaire, on note une augmentation des cellules en division. Les cellules, en voie de maturation, présentent plus de produits de sécrétion, à un stade plus précoce de leur cycle d'évolution, et semblent subir une hypertrophie plus rapide. Dans la zone pré-hypertrophique, la matrice intercellulaire présente des foyers de calcification précoce, ainsi que des fibres collagènes plus longues et plus épaisses que chez les témoins. Il apparait que, chez l'animal entier, des doses même élevées de testostérone provoquent initialement une stimulation de la prolifération chondrocytaire, avant de favoriser les processus de maturation.

     

Ultrastructural effects of estrogen on epiphyseal cartilage

Ten male weanling Holtzman rats, injected intraperitoneally with aqueous estradiol (Progynon, Schering), in daily doses of 1 μg. per g body weight, were sacrificed, simultaneously with controls receiving an equivalent amount of diluent, at intervals ranging from one hour to six days. Upper tibial epiphyseal cartilage plates (ECP), procesed for electron microscopy, revealed, as early as three hours after injection, appreciable enhancement of secretory activity, evidenced, in the zone of matrix secretion, by the abundance in Golgi cisternae of stippled material representing proteinpolysaccharide complexes. Disintegration of the lining membrane of individual Golgi vesicles was advanced after twenty-four hours; following three days of treatment, few vesicles remained intact, and pools of initially intravacuolar material were observable in the gound plasm. Long filaments, suggestive of primary or precursor collagen fibrils were apparent in this secretion. After six days, virtual lakes of this substance filled cells in the zone of prehypertophy, with consequent displacement of the rough endoplasmic reticulum against the cell periphery. Cytoplasmic vacuoles, containing mateerial similar to that found in the lacunar moat, and displaying finely beaded, radially arrayed filaments on the lining membrane were frequently encountered. Our observations suggest an initial acclleration of chondrocytic secretory activity, with subsequent retardation of transport. The resultant retention and intracellular polymerization of precollagenous products accelerates hypertrophy, thereby promoting early degeneration of chondrocytes. These ultrastructural alterations are apparently estrogen-specific.     

Fractures of the tibia through the proximal tibial epiphyseal cartilage

Thirty-nine fractures of the tibia involving the proximal tibial epiphyseal cartilage were treated at the Campbell Clinic over a twenty-five-year period. Stress roentgenograms were essential in making the diagnosis in three patients. Two patients had disruption of the popliteal artery, and both had posterior displacement of the tibial shaft. The other immediate complications that we encountered were anterior compartment syndrome, peroneal-nerve palsy, and associated ligamentous and meniscal injuries. Of twenty-eight fractures (twenty seven patients) with an average follow-up of 7.1 years, there were satisfactory results in twenty-four. Unsatisfactory results (four fractures) were due to chronic neurovascular insufficiency, growth disturbance, or traumatic arthritis.     

Electron probe analysis of cryosections of epiphyseal cartilage

Conventional methods for preparing specimens for analysis of the chemical composition of mineralizing tissues at the electron microscopic level can produce artefacts during fixation, dehydration, embedding and wet-sectioning. Cryoultramicrotomy provides a more reliable approach to electron probe analysis of initial apatite formation in calcifying cartilage. Two procedures are described (a) for a micro-analytical approach and (b) for a morphological study. By using a negative staining procedure (b) cytoplasmic details in terms of subcellular organelles and extracellular, membrane-bound, matrix vesicles could easily be identified in relatively large sections of epiphyseal cartilage. Unfixed, dry, unstained cryosections obtained by procedure (a) showed dense mineral granules (50–120 nm in diameter) in the mitochondria of chondrocytes and electron probe analysis of these gave a mean Ca/P mass ratio of 1.20. Adjacent cytoplasmic areas showed negligible levels of calcium. In the longitudinal septa matrix, vesicle-like particles could be identified with characteristic crystal needle clusters. Microanalysis of these structures showed them to have a mean Ca/P mass ratio of 2.15. Non-vesicular matrix appeared to be very low in calcium and phosphorus. The combination of cryoultramicrotomy and electron probe analysis of different areas in the same cryosection has provided some information on the coexistence of amorphous calcium phosphate in the mitochondria and of crystalline apatite in matrix vesicles a few micrometers away. These findings may thus explain the sequence of hydroxyapatite formation in epiphyseal cartilage.     

Lipid metabolism of chick epiphyseal bone and cartilage

The content and composition of lipids of chick epiphyseal tissue were investigated. The amounts of total lipids, triglycerides and phospholipids in bone were 7.50, 7.81 and 1.02 mg/g, respectively, and in cartilage, 5.20, 1.46 and 0.53 mg/g, respectively. The main fatty acids of both bone and cartilage were palmitic and oleic in phospholipids, and palmitic, oleic and linoleic in triglycerides. For the substrates studied, the order of incorporation into total lipids of both bone and cartilage was palmitate > glucose > acetate > citrate. Acetate was the main precursor for iatty acid synthesis whereas only a minor portion of glucose was found in the fatty acids. Esterification appeared to be the predominant pathway of lipid synthesis in chick bone and cartilage.

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Zusammenfassung Es wurde eine Untersuchung über den Gehalt und die Zusammensetzung der Lipide im Epiphysengewebe von Hühnchen durchgeführt. Es wurden 7,50 mg Gesamtlipide, 1,81 mg Triglyceride und 1,02 mg Phospholipide per Gramm Knochen gefunden. Die entsprechenden Werte per Gramm Knorpel waren 5,20, 1,46 und 0,53 mg. Die hauptsächlichsten Fettsäuren in den Phospholipiden von Knochen- und Knorpelgewebe waren Palmitin- und Ölsäure, in den Triglyceriden Palmitin-, Öl- und Linolsäure. Die untersuchten Verbindungen wurden in nachstehender Reihenfolge in Knochen- und Knorpel-Lipide eingebaut: Palmitin > Glucose > Acetat > Citrat. Acetat war die hauptsächliche Ausgangssubstanz für die Fettsäure-Synthese, während nur ein unbeträchtlicher Teil der Glucose in den Fettsäuren vorgefunden wurde. Veresterung ist anscheinend der vorherrschende Weg der Fettsynthese in Knorpel- und Knochengewebe von Hühnchen.

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Résumé Le contenu et la composition des lipides du tissue épiphysaire ont été étudiés chez le poulet. Les valeurs des lipides totaux, des triglycérides et des phospholipides sont, respectivement dans l'os, de 7,50, 1,81 et 1,02 mg/g et, dans le cartilage, de 5,20, 1,46 et 0,53 mg/g. Les principaux acides gras trouvés dans l'os aussi bien que dans le cartilage sont les acides palmitique et oléique, parmi les phospholipides, et les acides palmitique, oléique et linoléique parmi les triglycérides. Quant aux substrats étudiés, l'ordre d'incorporation dans les lipides totaux de l'os et du cartilage a été: palmitate > glucose > acétate > citrate. L'acétate était le principal précurseur dans la synthèse des acides gras, alors que seule une faible protion de glucose a été trouvée dans les acides gras. L'estérification semble donc être la voie prédominante de la synthèse des lipides dans l'os et le cartilage du poulet.

     

Localization of prostaglandin synthetase in chicken epiphyseal cartilage

Summary Prostaglandin synthetase activity in highspeed particulate fractions of chick epiphyseal cartilage has been characterized with respect to cofactor requirements, pH optimum, buffer-ion effects, types of prostaglandins formed, and the distribution of prostaglandin synthetase activity in zones of the epiphyseal plate.Direct homogenization of cartilage was found to be more efficacious than releasing chondrocytes by enzymatic digestion for preparation of prostaglandin synthetase, a homogenization time of 4 min yielding maximal activity. The optimal incubation medium contained 50 mM Tris buffer (pH 7.5), 2.5 mM epinephrine, 1 M hemoglobin, 3.25 mM glutathione, 200 g/ml enzyme protein, and 5 M substrate. Glutathione was effective only if present during homogenization. Rates of PGE₂ biosynthesis were linear up to 15 min and then rapidly declined, indicative of self-deactivation. The low levels of PGF₂ formed, and their decrease after 20 min incubation, suggests the possible presence of degradative enzymes.Prostaglandin synthetase was inhibited by aspirin, indomethacin, and vitamin E, but not vitamin K₁. Cation concentrations in the physiological range had only modest effects on prostaglandin biosynthesis, and then only if present during tissue homogenization. In the presence of phosphate buffer, Ca²⁺ was somewhat inhibitory. Since in the absence of phosphate Ca²⁺ had no deleterious effect, it is probably that the inhibitory effect was caused by precipitation of calcium phosphate.Hypertrophic and calcified cartilage exhibited significantly higher prostaglandin synthetase activity than the proliferating and maturing zones. The increased synthesis of prostaglandins in the low layers of the growth plate may indicate a role of these factors in chondrocyte differentiation and/or calcification.     

In vivo visualization of albumin degradation in the proximal tubule

Albuminuria is a key marker of renal injury and a major risk factor for cardiovascular disease. In vivo imaging techniques with fluorescent albumin have allowed visualization of its movement within the whole kidney but they could not distinguish between intact and degraded albumin. To visualize albumin degradation in proximal tubular cells in vivo we used an albumin conjugate (dye quenched (DQ)-albumin), which only fluoresces when it is degraded. In cultured proximal tubule cells, the fluorescent signal from DQ-albumin was dependent on endocytosis and lysosomal function and showed that at any time about 40% of endocytosed DQ-albumin was degraded. Significant accumulation of conventional Texas Red-labeled albumin and degraded DQ-albumin was found in rat proximal tubules 5 min after injection. Importantly, no hint of DQ-albumin was detected in the serum, suggesting that the fluorescent signal in the proximal tubules was derived from tubular degradation of intact albumin. Our study shows that DQ-albumin, together with conventional fluorescent conjugates of intact albumin, can be used to visualize albumin degradation by proximal tubules in vivo.     

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.