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Calcified Tissue Research

, Volume 14, Issue 1, pp 229–244 | Cite as

Histochemical studies on calcified tissues

I. Amino acid histochemistry of foetal calf and human enamel matrix
  • Mona M. Everett
  • William A. Miller
Original Papers

Abstract

Histochemical methods and dye binding studies were carried out on foetal calf tooth germsin situ fixed in cyanuric chloride or formalin. Protein concentration decreased upon maturation of enamel. The tissue was divided into two distinct bands, an outer region of newly formed enamel and an inner region of maturing enamel. What has previously been defined biochemically as mature enamel was not investigated. Histidine was present in enamel and easily iodinated but protected from benzoylation. Two methods for tyrosine analysis confirm its presence in both newly formed and maturing enamel but with some decrease upon maturation. The transition was gradual; Sulphydryl groups were present in newly formed, but not in maturing, enamel, with abrupt transition between the two zones. Tryptophane was demonstrated throughout the entire thickness of enamel. Arginine residues were not very reactive to Sakaguchi's method but they were demonstrable by fluorodinitrobenzene blockade of anionic dyes. Lysine was present, decreasing upon maturation. Carboxylic acid residues were demonstrated in both maturing and newly formed enamel but with distinct banding and noticeably fewer residues in maturing enamel. Anionic dye binding at various salt concentrations showed that the critical electrolyte concentration of enamel is moderately high and similar to that of keratin. Cationic dye binding studies indicated that ionization of carboxylic acid groups in newly formed enamel, but not maturing enamel, might be suppressed. Newly formed enamel has the histochemical characteristics of a keratin but maturing enamel does not, more nearly resembling one of a group of ectodermal globular proteins.

Key words

Teeth-enamel Histocytochemistry Calcification 

Résumé

Des études histochimiques et d'affinités tinctoriales sont menées sur des germes dentaires de foetus de veauxin situ, fixés dans du chlorure de cyanure ou du formol. La concentration en protéine décroit avec la maturation de l'émail. Le tissue est divisé en deux bandes distinctes: une région externe d'émail nouvellement formé et une région interne d'émail en maturation. L'émail mature, défini antérieurement par méthode biochimique, n'a pas été étudié. L'histidine, présente dans l'émail, est aisément traitée par l'iode: le traitement au benzol n'a pas d'effet. La tyrosine est mise en évidence par deux méthodes dans les deux types d'émail et décroit légèrement au cours de la maturation. La transition est progressive. Des groupements sulfhydriles sont présents dans l'émail jeune, mais non dans l'émail en voie de maturation, avec une transition nette entre les deux zones. Le tryptophane est présent dans toute l'épaisseur de l'émail. L'arginine ne réagit pas bien par la méthode de Sakaguchi, mais on peut la mettre en évidence en bloquant par la fluorodinitrobenzène les colorants anioniques. La lysine est présente et diminue avec la maturation. Les résidus d'acide carboxyliques sont mis en évidence dans les deux types d'émail, mais des bandes distinctes et moins de résidus sont observés au cours de la maturation. Les affinités tinctoriales anioniques à diverses concentrations de sels montrent que la concentration critique en électrolytes de l'émail est relativement élevée et semblable à celle de la kératine. Les affinités tinctoriales cationiques indiquent que l'ionisation des groupements d'acide carboxylique peut être supprimée dans l'émail jeune, mais non dans l'email en voie de maturation. L'émail jeune a les caractéristiques histochimiques d'une kératine, mais non pas l'émail mature, qui ressemble à un groupe de protéines globulaires ectodermiques.

Zusammenfassung

Histochemische Methoden und Farbbindungs-Untersuchungen wurden an Zahnkeimen von Kalbsembryonen in situ, welche in Cyanurchlorid oder Formalin fixiert worden waren, ausgeführt. Die Proteinkonzentration nahm mit dem Reiferwerden des Schmelzes ab. Das Gewebe wurde in zwei genau abgegrenzte Bänder unterteilt, einen äußeren Bereich aus neu gebildetem Schmelz und einen inneren Bereich aus reifendem Schmelz. Was früher biochemisch als reifer Schmelz definiert wurde, wurde nicht untersucht. Histidin lag im Schmelz vor und konnte leicht iodiert werden, eine Benzoilierung fand jedoch nicht statt. Zwei verschiedene Tyrosinanalysen bestätigen dessen Gegenwart in neugebildetem und reifendem Schmelz, wobei es aber beim Reifeprozess etwas abnahm. Der Übergang zeigte sich fließend. Sulphydryl-Gruppen waren in neugebildetem, aber nicht in reifendem Schmelz vorhanden, wobei sich ein abrupter Übergang zwischen den beiden Zonen fand. Tryptophan konnte durch die gesamte Schmelzbreite nachgeweisen werden. Argininrückstände reagierten kaum auf die Sakaguchi-Methode, aber sie konnten durch die anionische Farbstoffe blockierende Fluorodinitrozenzen-Methode nachgewiesen werden. Lysin war anwesend und nahm im Laufe des Reifeprozesses ab. Carboxylsäure-Rückstände wurden in reifendem und neugebildetem Schmelz nachgewiesen, jedoch mit scharfer Abgrenzung und deutlich weniger Rückständen in reifendem Schmelz. Die Bindung mit anionischen Farbstoffen bei verschiedenen Salzkonzentrationen zeigte, daß die kritische Elektrolyten-Konzentration nicht besonders hoch und derjenigen von Keratin ähnlich ist. Untersuchungen mit kationischen Farbstoffen deuteten an, daß die Ionisierung von Carboxylsäure-Gruppen in neugebildetem, nicht aber in reifendem Schmelz, unterdrückt werden kann. Neugebildeter Schmelz hat die histochemische Eigenschaften von Keratin; reifender Schmelz hingegen hat diese Eigenschaft nicht und gleicht eher einem Vertreter der Gruppe der ektodermalen globulären Proteine.

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Copyright information

© Springer-Verlag 1974

Authors and Affiliations

  • Mona M. Everett
    • 1
  • William A. Miller
    • 1
  1. 1.Department of Oral BiologyState University of New York at BuffaloBuffaloUSA

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