Embryonic chick mandibles (Hamburger and Hamilton [HH] stages 17-25) were cultured in the presence of various concentrations of vitamin A to determine the effect of hypervitaminosis A on membrane bone formation. In normal development, the mandible differentiates a centrally located Meckel's cartilage surrounded by membrane bones. Mandibles cultured without added vitamin A differentiated normally, though the timing of differentiation was retarded from that in ovo. Treatment with vitamin A interfered with skeletogenesis to varying degrees depending upon the initial age of the explant and the concentration of vitamin A. At low concentrations of vitamin A (1 pgiml), neither cartilage nor membrane bone formed in young explants (HH stage 171, whereas cartilage formed in 78% and membrane bone in 11% of older explants (HH stage 25). Higher concentrations of vitamin A (2-4 pg/ml) inhibited membrane bone formation in all explants, and 4 pgi ml of vitamin A inhibited chondrogenesis in most (88%) of the older explants. To determine whether tissue interactions influence this effect of vitamin A on skeletogenesis, mandibular mesenchyme was separated from its epithelium and treated with vitamin A. Under normal culture conditions, isolated mesenchyme (HH stage 25) differentiated both cartilage and membrane bone. Hypervitaminosis A inhibited membrane bone formation in the isolated mesenchyme at all levels tested (1-4 pgi ml) and inhibited chondrogenesis at levels 2-4 pgiml. Hence, vitamin A can act directly upon the mesenchyme to inhibit both membrane bone formation and chondrogenesis, but its action is mitigated by the presence of the epithelium.
Culturing procedures
Tissues were cultured on Millipore (white, 0.45-pm porosity, 5 mm diam., 125-150 pm thick; Millipore Filter Corp., Bedford, MA) and Metricel filter disc (white, 5 mm diam., 0.20 pm porosity, 150 pm thick; Gelman Science Inc., Ann Arbor, MI), supported by stainless ~