Abstract
A method is described employing 95% hydrazine which completely deproteinates and slightly dehydrates bone under nearly anhydrous conditions with only moderate heating. This method induced only minor chemical changes and no alterations in structural properties of the mineral phase. Physicochemical data are presented demonstrating that although rat bone crystals more closely resemble synthetic controls made in carbonate-rather than hydroxide-rich media, rat bone apatite cannot be interpreted in terms of known or postulated crystal models in any meaningful fashion. CO 2−3 infrared band assignments made from spectra of whole bone are shown to be in error due to the presence of protein absorption bands. Absorotion of HPO 2−4 was observed in infrared spectra of young rat bone mineral. Detailed X-ray diffraction comparisons of deproteinated rat bone before and after hydrolysis clearly demonstrated the presence of amorphous calcium phosphate. Electron microscopy indicated that very small apatite crystals were present in rat bone which might also contribute to the overall mineral pool amorphous to X-ray diffraction. Electron microscopy also showed domains in rat bone mineral where plate-like apatite crystals maintained a netc-axis orientation despite the removal of their fibrous matrix.
Résumé
Une méthode, utilisant 95% d'hydrazine, permet de déprotéiniser et de déshydrater légèrement l'os dans des conditions presqu'anhydres, avec une élévation de température modérée. Cette méthode ne provoque que des modifications chimiques mineures, sans altération des propriétés structurales de la phase minérale. Les résultats physico-chimiques démontrent que bien que les cristaux d'os de rat sont viosins de cristaux synthétiques témoins constitués dans des milieux, riches en carbonate plutôt qu'en hydroxyde, l'apatite osseux de rat ne parait pas analogue à des modèles cristallins connus ou imaginés. Des déterminations de bande infra-rouge CO 2−3 , réalisées à partir de spectre d'os total, semblent faussées par la présence de bandes d'absorption protéique. L'absorption d'HPO 2−4 est étudiée à l'aide de spectres infra-rouges de minéral osseux de jeunes rats. Des comparaisons détaillées en diffraction par raysons X d'os déprotéinisé de rats, avant et après hydrolyse, démontrent nettement la présence de phosphate de calcium amorphe. La microscopie électronique indique que de petits cristaux d'apatite dans l'os de rat sont susceptibles de contribuer au pool minéral amorphe en diffraction en rayons X. La microscopie électronique montre des plages de minéral osseux de rat où des cristaux d'apatite en forme de plaque, présentent une maille cristalline avec axe C malgré l'élimination de leur matrice fibreuse.
Zusammenfassung
Es wird eine Methode beschrieben, wobei durch Anwendung von 95% Hydrazin ohne Wasserzugabe und mit nur geringem Erhitzen dem Knochen das gesamte Protein und ein kleiner Teil des Wassers entzogen wird. Diese Methode führte nur zu geringen chemischen Veränderungen und veränderte die strukturellen Eigenschaften der Mineralphase in keiner Weise. Physikochemische Daten wurden erbracht, welche zeigen, daß — obwohl die Kristalle von Rattenknochen den synthetischen Kontrollen (in Karbonat- und nicht hydroxydreichen Medien hergestellt) eher gleichen — Apatit aus Rattenknochen nicht auf sinnvolle Weise mittels bekannten oder postulierten Kristallmodellen interpretiert werden kann. CO 2−3 -Infrarotbandenzuteilungen, welche von Spektren aus dem Gesamtknochen gemacht wurden, geben wegen der Anwesenheit von Proteinabsorptionsbändern falsche Resultate. Die Absorption von HPO 2−4 wurde in den Infrarotspektren von Knochenmineral aus jungen Ratten beobachtet. Ein Vergleich der detaillierten Röntgendiffraktion von deproteinisiertem Rattenknochen vor und nach der Hydrolyse wies deutlich auf die Anwesenheit von amorphem Calciumphosphat hin. Die Elektronenmikroskopie zeigte kleine Apatitkristalle im Rattenknochen, welche zum Gesamtmineralpool beitragen könnten, der bei der Röntgendiffraktion amorph ist. Die Elektronenmikroskopie zeigte auch Gebiete im Rattenknochenmineral, wo plättchenartige Apatitkristalle eine deutlichec-Achsenorientierung beibehielten, obwohl ihre fibröse Matrix entfernt worden war.
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Termine, J.D., Eanes, E.D., Greenfield, D.J. et al. Hydrazine-deproteinated bone mineral. Calc. Tis Res. 12, 73–90 (1973). https://doi.org/10.1007/BF02013723
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DOI: https://doi.org/10.1007/BF02013723