Optimization of perfusion decalcification for bones and joints in rats

Abstract

Decalcification of osseous tissues by perfusion of decalcifying solution into the vascular system has never been applied to the study of peripheral joints. To optimize perfusion methods, rats were decalcified by direct immersion or by one of two perfusion techniques: 1) systemic perfusion circulating the decalcifying solution from the ascending aorta; and 2) regional perfusion circulating the solution to the lower extremities from the abdominal aorta. The process of decalcification was monitored by serial radiographic examinations. After decalcification, bone and joint samples were stained for histochemistry and immunohistochemistry. With systemic perfusion, the decalcification time, dependent on weight, was markedly reduced compared to immersion. Regional perfusion decalcification was faster than all other methods studied. Microstructural preservation was comparable and immunostaining quality often improved. Applications of this work will improve the study of basic skeletal and articular problems. Anat Rec 260:222–227, 2000. © 2000 Wiley‐Liss, Inc.

Plastination of decalcified bone by a new resin technique

“…(The needle had to pass through the bone without any force). [ 13 ] Decalcification was investigated by X-ray radiology for each bone after decalcification as a standard method. [ 14 15 16 ] In this step, all of the human bones and half of the sheep femur bones were decalcified.…”

“…For neutralizing the remained acid, the specimens were put in 5% sodium sulfate solution for 24 hours. [ 12 13 14 ]…”

Methodology

“…Detail of trabecular bone matrix decalcified before embedding by prolonged immersion in EDTA: the collagen fibrils are highly dissociated. Uranyl acetate and lead citrate, ×54,000 immersion methods, especially when the technique adopted is regional perfusion by circulating the solution in the lower extremities from the abdominal aorta (Trudel et al 2000). Incidentally, perfusion of a decalcifying solution can be used to achieve decalcification of the whole skeleton (Trudel et al 2000).…”

“…Uranyl acetate and lead citrate, ×54,000 immersion methods, especially when the technique adopted is regional perfusion by circulating the solution in the lower extremities from the abdominal aorta (Trudel et al 2000). Incidentally, perfusion of a decalcifying solution can be used to achieve decalcification of the whole skeleton (Trudel et al 2000). Buffered 4% formaldehyde or 2% glutaraldehyde, possibly followed by osmium tetroxide postfixation, are the most frequently used perfusion fixation mixtures (Reale and Luciano 1970;McKee et al 1991).…”