Chondrocyte and osteoblast differentiation stage-specific monoclonal antibodies as a tool to investigate the initial bone formation in developing chick embryo.

Taking advantage of monoclonal antibodies raised against chick hypertrophic chondrocytes (BD5, LA5, AD2) and osteoblasts (SB1, SB2, SB3, SB5), we have investigated expression of differentiation stage-specific antigens by further differentiating hypertrophic chondrocytes. Expression of all antigens was developmentally regulated during in vitro further differentiation of hypertrophic chondrocytes and all monoclonal antibodies, including those raised against osteoblasts, recognized antigens synthesized by chondrocytes at specific differentiation stages. In particular, the expression of the SB5 antigen, considered specific for secretory osteoblasts incorporated into the bone matrix, progressively increased during the chondrocyte culture and reached its maximum at the time of matrix mineralization. Interestingly, the SB3 antigen was transiently expressed at an intermediate phase of the culture with a staining pattern characteristic of cell-cell adhesion proteins. When cryosections of the chondro-bone junction of developing embryo tibiae were stained, it was observed that monoclonal antibodies raised against hypertrophic chondrocytes stained frank hypertrophic and elongated chondrocytes (BD5, LA5), a very discrete peripheric layer of cartilage (AD2), and cells embedded in the initial osteoid matrix (LA5), but failed to stain cells in the preosteoblast and osteoblast region. On the contrary, monoclonal antibodies recognizing antigens on the surface of cells in the osteogenic layer surrounding the initial bone, also stained frank hypertrophic and/or elongated chondrocytes on the cartilage side of the initial bone (SB1, SB2, SB3). The SB5 monoclonal antibody, in addition to staining cells in the osteoid, also stained a few chondrocytes immediately adjacent. These observations support the concept that hypertrophic chondrocytes may further differentiate to osteoblast-like cells and participate to the initial bone formation.