An Infiltration and Clearing Technic to Reveal the Vascular Pattern of Ethylenediamine-Treated Cortical Bone

Abstract

The vascular canals of cortical bone are revealed by: (1) removing all organic tissue with ethylenediamine (ed), (2) infiltrating the Haversian and Volkmann canals with a pigment and (3) finally clearing the specimens in styrene. Buccal and lingual cortical plates of mandibles were freed of spongy bone and placed in a Soxhlet extractor. Extraction was carried out with a solution consisting of 4 volumes of ed and 1 volume of distilled water which was maintained at a temperature of 118°C for 24 hr. After extraction, the organic-free specimens were washed in water for 1 hr and dried. The dried bone was immersed in a 25% India ink suspension and placed in a vacuum chamber until bubbling ceased. The bones were then dried and the surface carbon was removed with a CO₂-driven stream of dolomite powder from an Airdent unit. Compressed air was used to remove the residual dolomite powder from the specimens. They were then immersed in styrene until clear. The specimens could be studied directly from the styrene, since the inorganic portion of the bone became transparent and carbon retained on the walls of the vascular channels within the calcified tissue was clearly visible. Plastic can be substituted for styrene for infiltrating the spaces within the specimen which were previously occupied by organic material. After infiltration and curing of the plastic, the embedded specimens can be decalcified with concentrated HCl. After decalcification, the voids created by the removal of the inorganic matrix can then be filled with plastic. Substituting plastic for areas previously occupied by both the organic and inorganic elements results in an easily visualized, carbon-black three-dimensional preparation of the vascular pattern of bone.