EXPRESSION OF OSTEOPONTIN IN MECKELS CARTILAGE CELLS DURING PHENOTYPIC TRANSDIFFERENTIATION IN-VITRO, AS DETECTED BY IN-SITU HYBRIDIZATION AND IMMUNOCYTOCHEMICAL ANALYSIS

The localization of osteopontin (OP) was examined in Meckel's cartilag e cells that bipotentially expressed cartilage and bone phenotypes dur ing cellular transformation in vitro. Cultured cells were analyzed by in situ hybridization, immunostaining followed by light and electron m icroscopy, electron microscopy, and electron probe microanalysis. The combination of ultrastructural analysis and immunoperoxidase staining indicated that OF-synthesizing cells were cells that were autonomously undergoing a change from chondrocytes to bone-forming cells at the to p of nodules. Double immunofluorescence staining of 2-week-old culture s revealed that OP was first synthesized by chondrocytic cells at the top of nodules. After further time in culture, the distribution of OP expanded from the central toward the peripheral regions of the nodules . Electron probe microanalysis revealed that the localization of OP wa s associated with matrices of calcified cartilage and osteoid nodules that contained calcium and phosphorus. Immunoperoxidase electron micro scopy revealed that, in addition to the intracellular immunoreactivity in chondrocytes and small round cells that were undergoing transforma tion, matrix foci of calcospherites and matrix vesicles, in particular , included growing crystals that were immunopositive for OF. An intens e signal due to mRNA for OP in 3-week-old cultures was detected in nod ule-forming round cells, while fibroblastic cells, spreading in a mono layer over the periphery of nodules, were only weakly labeled. These f indings indicate that OP might be expressed sequentially by chondrocyt es and by cells that are transdifferentiating further and exhibit an o steocytic phenotype, and moreover, that expression of OP is closely as sociated with calcifying foci in the extracellular matrix.