IN-SITU LEVELS OF INTRACELLULAR CA2-PLATE CARTILAGE( AND PH IN AVIAN GROWTH)

Interactions with the extracellular matrix, accumulation of Ca2+, form ation of matrix vesicles, and regulation of tissue pH by growth plate chondrocytes all appear to be vital to endochondral calcification. Thu s, the activities of Ca2+ and H+ ions in these cells, while still embe dded in their organic matrix, are of great interest, Using laser confo cal imaging and sensitive Ca2+ (Indo 1) and pH (BCECF) probes, cellula r Ca2+ and pH were analyzed in thin sections of freshly isolated carti lage, Mean values of cytosolic Ca2+ in cells from the various zones of the growth plate were quite similar but levels in individual cells an d subcellular compartments varied significantly, Ca2+ was elevated int ensely near the periphery of cells in the zones of maturation and hype rtrophy, and many Ca2+ rich particles were seen in the matrix near the se cells. Levels of Ca2+ within the cells varied with time, In the pro liferative region, cyclical increases and decreases in Ca2+ were seen, but there was little shedding of Ca2+ rich particles, However, after repeated Ca2+ cycling, in the zones of maturation and hypertrophy, Ca2 + rich particles were shed from the cell surface, forming what appeare d to be matrix vesicles, Intracellular pH levels also varied significa ntly within the chondrocytes and between the cells and zones, Numerous focal elevations in pH (> 8,0) were seen in central regions of the ma turing and early hypertrophic cells, with lower pH (6.5-7.2) near the cell periphery of the late hypertrophic and calcifying cells, This pat tern of cytoplasmic alkalinization and subsequent acidification appear s to contribute to loading of Ca2+ and Pi into matrix vesicles during their formation bg the chondrocytes.