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Calcified Tissue Research

, Volume 4, Issue 1, pp 20–38 | Cite as

A zonal analysis of inorganic and organic constituents of the epiphysis during endochondral calcification

  • Roy E. Wuthier
Original Papers

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.

Key words

Calcification Epiphyseal Cartilage Bone Electrolytes Organic matrices 

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.

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.

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Copyright information

© Springer-Verlag 1969

Authors and Affiliations

  • Roy E. Wuthier
    • 1
  1. 1.Forsyth Dental CenterBoston

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