Calcification. VII. Reversible inactivation of calcification in vitro and related studies.

Calcification in vitro of the hypertrophic epiphyseal cartilage has served as a useful indicator in the study of the “local factors” involved in calcification (12, 13, 25, 29-31, 34, 37, 38, 40, 44, 54). The appearance of new calcification depends on the functioning of the complete system essential for mineralization. The histological appearance of calcification in vitro of the hypertrophic epiphyseal cartilage resembles that obtained in calcification in viva. New mineral deposits occur in the matrix and have a honeycomb-like structure (31, 39). X-ray diffraction studies have shown that the new mineralization obtained has the same lattice for calcification in vitro as that found for calcification in vivo, viz. apatite (49). Most of our knowledge of the “local factors” concerned with calcification comes from such in vitro studies. Thus, Robison (30, 31,33) has been led to stress the importance of phosphatase. Gutman et al. (12,13) and Marks and Shorr (23) have presented evidence that phosphorylative glycogenolysis is an integral part of the calcification process. Yet one can demonstrate the presence of phosphatase and the glycogenolytic cycle in tissues other than bone which do not calcify. In rickets due to strontium, calcification in vitro in the presence of small amounts of magnesium is strongly inhibited without a concurrent decrease in phosphatase activity (4446, 48). Waldman (54) has claimed that calcification in vitro can take place when phosphatase is inactivated. Siffert (41) reported that phosphatase activity is associated with the preosseous tissue and not with the process of calcification. Paff and Salhnan (26) found that calcification can be stopped in a tissue culture by means of alizarin S, without inhibiting phosphatase and without removing calcium and phosphate ions to a significant degree. From these considerations one may perceive that the r&le in calcification of those enzymes concerned with the disposition of phosphate ion has not