Objectives: First, to continue studying the mechanism of cartilage and bone induction by FL, HeLa and other cultured human malignant cells. Previous studies have shown that bone induction requires: 1) suppression of the immune response in recipient mice with cortisone or ALS; 2) injection of viable human cells and 3) injection into skeletal muscle. In the proposed experimental we shall attempt to explant pre- osseous thigh tumors to mouse brain (a site capable of supporting tumor growth and differentiation); to organ culture (where recent studies indicate the development or an "osteoid"-like matrix after 7 days); and to chick chorioallantoic membranes which is known to support growth and differentiation of xenogenic grafts. The differentiation of cartilage and bone will be determined by microscopy using stains for metachromasia and autoradiography for Ca45. Electron microscopy will be used to determine the presence of proteoglycan granules and calcifying matrix vesicles which are characteristic of cartilage. The accumulation of proteoglycans and calcium will be determined biochemically. When a model system is established, in which cultured human cells and susceptiible mouse cells can be interacted to produce bone, we shall attempt to further characterize the mechanism of bone induction. Second, to continue studying the structure, composition and mineralizing functions of calcifying matrix vesicles which we originally identified in FL-induced cartilage, and subsequently were shown to play a role in calcification of such diverse tissues as developing bone, dentin, normal growth cartilage and, recently, in calcifying human aortic valves and aortas during arteriosclerosis. We shall attempt to study, 1) The vesicle calcification mechanisms by observing and experimentally attempting to alter the uptake of calcium into vesicle- containing intact cartilage or bone, and into isolated matrix vesicles, and 2) The manner of vesicle formation using autoradiography with H3- glycerol or other lipid substituents which are concentrated into the membranes of precursor cells, and thence into the matrix.