REGULATED PRODUCTION OF MINERALIZATION-COMPETENT MATRIX VESICLES IN HYPERTROPHIC CHONDROCYTES

Matrix vesicles have a critical role in the initiation of mineral depo sition in skeletal tissues, but the ways in which they exert this key function remain poorly understood. This issue is made even more intrig uing by the fact that matrix vesicles are also present in nonmineraliz ing tissues. Thus, we tested the novel hypothesis that matrix vesicles produced and released by mineralizing cells are structurally and func tionally different from those released by nonmineralizing cells. To te st this hypothesis, we made use of cultures of chick embryonic hypertr ophic chondrocytes in which mineralization was triggered by treatment with vitamin C and phosphate. Ultrastructural analysis revealed that b oth control nonmineralizing and vitamin C/phosphate-treated mineralizi ng chondrocytes produced and released matrix vesicles that exhibited s imilar round shape, smooth contour, and average size. However, unlike control vesicles, those produced by mineralizing chondrocytes had very strong alkaline phosphatase activity and contained annexin V, a membr ane-associated protein known to mediate Ca2+ influx into matrix vesicl es. Strikingly, these vesicles also formed numerous apatite-like cryst als upon incubation with synthetic cartilage lymph, while control vesi cles failed to do so. Northern blot and immunohistochemical analyses s howed that the production and release of annexin V-rich matrix vesicle s by mineralizing chondrocytes were accompanied by a marked increase i n annexin V expression and, interestingly, were followed by increased expression of type I collagen. Studies on embryonic cartilages demonst rated a similar sequence of phenotypic changes during the mineralizati on process in vivo. Thus, chondrocytes located in the hypertrophic zon e of chick embryo tibial growth plate were characterized by strong ann exin V expression, and those located at the chondro-osseous mineralizi ng border exhibited expression of both annexin V and type I collagen. These findings reveal that hypertrophic chondrocytes can qualitatively modulate their production of matrix vesicles and only when induced to initiate mineralization, will release mineralization-competent matrix vesicles rich in annexin V and alkaline phosphatase. The occurrence o f type I collagen in concert with cartilage matrix calcification sugge sts that the protein may facilitate crystal growth after rupture of th e matrix vesicle membrane; it may also offer a smooth transition from mineralized type II/type X collagen-rich cartilage matrix to type I co llagen-rich bone matrix.