Abstract. Conditions have been defined for promoting growth and differentiation of hypertrophic chondrocytes obtained in culture starting from chick embryo tibiae . Hypertrophic chondrocytes, grown in suspension culture as described (Castagnola P., G. Moro, F. Descalzi Cancedda, and R. Cancedda . 1986. J. Cell Biol. 102 :2310-2317, when they reached the stage of single cells, were transferred to substrate-dependent culture conditions in the presence of ascorbic acid. Cells showed a change in morphology, became more elongated and flattened, expressed alkaline phosphatase, and eventually mineralized . Type II and X collagen synthesis was halted and replaced by type I collagen synthesis . In addition the cells started to produce and to secrete in large amount a protein with an apparent molecular mass of 82 KD in reducing conditions and 63 KD in unreducing conditions. This protein is soluble in acidic solutions, does not contain oNG bone organogenesis occurs in the embryo by endochondral ossification from undifferentiated mesenchyme. During the early stages of development, mesenchymal cells in the limb buds condense to form a core of differentiated chondrocytes ; osteogenesis starts at the periphery ofthe cartilage core, which is subsequently invaded by blood vessels and replaced by bone marrow and trabecular bone. After birth, similar events take place in the long bone growth plate and at the bone fracture sites. Bone formation and remodeling have been extensively investigated, starting from pioneering work describing the morphological and biochemical changes occurring during early bone formation to more recent studies aimed at the elucidations of the cellular and molecular mechanisms involved (7,22,34) . It is widely agreed that cells present in a continuous collar surrounding, but separated from the cartilage rudiment, give rise to osteoblasts, i.e., cells responsible for the synthesis and mineralization of the osteoid extracellular matrix . In the past, occasionally and recently more frequently, it has been postulated thatgrowth platehypertrophic chondrocytes might also contribute to the formation of a bone matrix, since in some organ culturesthese cells start to express bone markers . During culture of mouse mandibular condyles, the expression of type I collagen, osteonectin, alkaline phosphatase, osteopontin, and osteocalcin by mature chondrocytes was detected by in situ hybridization (38). A morphological study © The Rockefeller University Press, 0021-9525/92/04/427/9 $2 .00 The Journal of Cell Biology, Volume 117, Number 2, April 1992 427-435 collagenous domains, and is glycosylated . The Ch21 protein, a marker of hypertrophic chondrocytes and bone cells, was synthesized throughout the culture . We have defined this additional differentiation stage as an osteoblast-like stage . Calcium deposition in the extracellular matrix occurred regardless of the addition of ß glycerophosphate to the culture medium . Comparable results were obtained both when the cells were plated at low density and when they were alrea...

During osteogenesis, in vitro, of tibial-derived rat osteoblasts (ROB) and derived clones, changes occur in the interactions of mature osteoblasts with the endogenous extracellular matrix (ECM) and these culminate in the formation of tridimensional nodules, which become sites of mineral deposition. We investigated if these changes might be mediated by remodeling of ECM, and we focused our study on the neutral metalloproteinases (MMPs), known agents of matrix remodeling, and on their tissue inhibitors (TIMPs). We report that during in vitro differentiation, osteoblasts express the secreted MMP-2 and -9 and the membrane gelatinase MMP-14.

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This study describes the adhesion of human osteoblasts, cultured in vitro, to proteins of the extracellular matrix, the biosynthesis of integrins, their topography and organization in focal contacts. The adhesion of osteoblasts to laminin, type I collagen, vitronectin and fibronectin was 77-100%, in 2 h and at 55 nM substrata concentration, and it was accompanied by spreading of the cells. Adhesion to fibronectin (FN), laminin (LN) and type I collagen (COL) was inhibited by antibodies to the beta 1 integrin and antibodies to the alpha 5 chain affected adhesion only to fibronectin. Using a panel of polyclonal antibodies against alpha 2, alpha 3, alpha 5, alpha v, beta 1 and beta 3 integrins we detected synthesis of alpha 3 beta 1, alpha 5 beta 1, alpha v beta 3, and an alpha v beta 1-like dimer by immunoprecipitation of metabolically labelled cell lysates. Studies of immunolocalization demonstrated the presence of the same integrins identified in lysates, plus alpha 4, alpha 1 and beta 5 subunits. In cells adhering in the presence of serum we showed organization of beta 3 and alpha v integrins in focal contacts. In cells adhering to fibronectin alpha 5 and beta 1 integrins were localized in focal contacts. In cells spread on laminin or type I collagen none of the integrins investigated was localized in focal contacts.