Electron microscope comparison of freeze-substitution and conventional chemical fixation of undecalificied human dentin.

Within the stated limits of cell fine structure preservation and within the observed anatomical limits specified in the Results and Discussion sections, a freeze-substitution method using an ethylene glycol-Hank's solution eutectic mixture with a glutaraldehyde additive can be used to effectively prepare undecalcified human dentin for electron microscopy. The ultrastructual appearance of the odontoblast cell body and the odontoblastic process subjected to freeze-substitution differs from that seen with conventional chemical fixation. Artifacts produced by freeze-substitution differ in appearance and frequency of occurrence from those produced by glutaraldehyde-osmium tetroxide sequential double fixation. The cellular component of dentin shows greater structural preservation of protein when it is subjected to freeze-substitution that when it is prepared by conventional chemical fixation. The absence of ice crystal defects in the odontoblastic process in calcified dentin and the presence of ice crystal defects in the odontoblast cell body suggest that intracellular water in the odontoblastic process in the calcified dentin may exist in a more highly structured state than intracellular water in the odontoblast cell body. If intracellular water exists in a more highly structured state in the odontoblastic process of the calcified dentin than in the cell body, the ratio of protein molecules to cytoplasmic volume may be greater in the odontoblastic process than in the cell body. After glutaraldehyde-osmium tetroxide sequential double fixation, the use of graded alcohol dehydration obtained cell fine structure preservation and artifact control superior to that obtained by use of ethylene glycol cryodehydration. Further refinements of the freeze-substitution technique, as it applies to the preparation of undecalcified human dentin, are necessary to increase the amount of cellular preservation, to decrease the number of ice crystal artifacts, and to improve the overall quality of cell fine structure preservation.