Comparison of the Effects of Papain and Vitamin A on Cartilage. II. The Effects on Organ Cultures of Embryonic Skeletal Tissue

The effects of papain protease and of vitamin A on explanted limb bone rudiments from 7- and 13-day chick embryos and fetal mice have been studied and compared. The incubation of cartilaginous rudiments from 7-day chick embryos in a solution containing papain and cysteine resulted in complete loss of the metachromasia of the cartilage matrix within 1 hour; explants treated in this fashion recovered normal metachromatic staining properties when grown in normal medium for 4 days. The incubation of 7-day chick cartilage rudiments in a solution containing papain without cysteine resulted in partial loss of metachromasia from cartilage within 1 hour; the addition of vitamin A to the solution did not enhance the effect of papain during this period. The addition of papain to the culture medium in which 7-day chick embryo cartilage rudiments were grown resulted in uniform loss of the metachromasia of the cartilage matrix; similar explants grown in the presence of excess vitamin A also showed loss of the metachromasia of cartilage, but certain regions of the cartilage were affected earlier and more severely than others. Changes in cartilage cells, including loss of glycogen, occurred when the rudiment was grown in medium containing excess vitamin A, but not when it was grown in the presence of papain. Bone rudiments from 13-day chick embryos showed changes in cartilage similar to those seen in 7-day chick embryo rudiments when grown in the presence of papain or of excess vitamin A; the existing bone was not affected under these conditions. When grown in the presence of papain or excess vitamin A, the cartilage of late fetal mouse bone underwent changes similar to those already described in chick embryo rudiments. In contrast to the chick embryo rudiments, those from the fetal mouse showed rapid resorption of bone when grown in the presence of excess vitamin A. Papain had no effect on bone from either source. The changes seen in cartilage of explants grown in the presence of vitamin A and papain together were greater than those seen with either agent alone. The changes seen in fetal mouse bone grown in the presence of vitamin A were not enhanced by the additional presence of papain. On the basis of these observations, it is suggested that the changes in cartilage seen in experimental hypervitaminosis A may be the result of activation of a proteolytic enzyme or enzymes with properties similar to papain.