Calcium Deposition in the Forelimbs of the Chick Embryo

The osteogenesis of the appendicular skeleton of the chick embryo has been carefully considered for a number of years. The distinction between perichondral ossification, the first to appear, and endochondral ossification was pointed out clearly by Lillie ('19). The first appearance of calcification in the endochondral ossification was stated by Lillie to be at 8 to 9 days in the clavicle of the chick embryo. This bone is considered the first ossification center of the body. The endochondral ossification with deposition of calcium begins quite late when the cartilage is removed by osteoclasts, that is, in the 17 to 18-day embryo. The same problem was taken up again by Fell ('25) and some years later by Fell and Robinson ('34). They gave the age of the embryo ;it which calcification first appears as between the 7th and 8th days of embryonic development. According to their investigations, carried out with the von Kossa silver nitrate method (von Kossa, '01; see also Lison, '36) , by the end of the 8th day the calcified region covers about one fifth of the total length of rudimentary bone; at either end a considerable part of the osteoid sheath remains uncalcified. The deposition of calcium salts was much later reconsidered by Paff et al. ('51 ) in tissue culture, studied by the use of alizarin dye. The peripheral zone of the bone was again the first to show ossification; the formation of the first spicules occurred several days later. This alizarinsensitive zone might or might not be present in fibulas of the 8-day-old chick embryo. Some years later Johnston and Comar ( ' 5 5 ) , investigating the contribution of calcium from the albumen, yolk and shell to the developing chick embryo with calcium4j, mentioned the increase of ash weight during the first 8 to 9 days of incubation. This was explained as due to a preossification period in which cdcium salts were accumulated, preceding the true ossification stage between the 10th and 19th days of development. This work was followed by the autoradiographic research of Johnston ('58) on the deposition of calcium in long bones of chick embryos. According to the experiments of this author, the osteogenetic sequence begins at about the 7th to 8th day with a sheath of osteogenic fibers formed beneath the perichondrium and with a negligible amount of calcium present. No formation of bone salts was demonstrable using the von Kossa technique. Only later does calcium increase and become confined very largely to a layer beneath the periosteum leading to the formation of a primary lamella which seems to be essentially acellular. This present work will attempt to present a study of the early presence of calcium as bone salts, in the forelimbs of the chick embryo, utilizing the alizarin red S technique in sections. This dye was chosen, bccause it had been used successfully in histochemical studies on other material. McGee-Russell ('58 ) , in his thorough analysis of the histochemical methods for calcium, considered sodium alizarin sulphonate the best anthraquinone dye. Hjs investigations showed that this dye was superior to the other staining solutions such as hematoxylin and it was more specific than the von Kossa technique as well as the other methods based on the conversion of calcium salts into salts of other metals. -