Mineralization during Long-Term Cultivation of Chick Embryos in Vitro

Abstract

Summary

An experimental model is described which permitted the long-term and continuous cultivation of shell-less chick embryos in petri dishes from 0 to 19 days in vitro. This procedure permits the study of development, skeletal morphogenesis and mineralization during embryogenesis in the absence of the egg shell. Embryos cultivated in Dulbecco's (calcium-magnesium-free) phosphate-buffered saline developed in petri dishes were similar to normal chick embryos incubated in situ, albeit somewhat smaller in overall size. Congenital malformations of the beak, limbs, or cranium were rarely detected in embryos cultured in vitro. Light microscopic and ultrastructural characteristics of chondrogenesis and osteogenesis among developmentally staged, shell-less embryos and controls were remarkably similar. In situ and in vitro mineralization of femur bone development was initially detected in stage-34 embryos and examined until embryos reached stage 42 (19 days of incubation). The calcium in the chick embryo has a dual origin: Experiments by Johnston and Comar show that prior to stage 36 most of the calcium originates in the yolk (8), whereas after stage 38 calcium ions are rapidly translocated from the egg shell to the chick skeletal system (8, 11, 18, 22). In the absence of the egg shell during in vitro chick embryogenesis, mineralization is regulated and sustained using calcium present in the yolk.

The authors wish to thank Mrs. Peggy Meek for her assistance in typing the manuscript. This research was supported by NIH Grants DE-02848-09, DE-03569-07, and DE-07006-04, U.S.P.H.S.