Localization of endogenous osteocalcin in neonatal rat bone and its absence in articular cartilage: Effect of warfarin treatment

Abstract

Immunocytochemistry after cryoultramicrotomy was used to localize endogenous osteocalcin in bone (calvaria, femoral diaphysis) and epiphyseal femoral cartilage from 8-day-old rats treated (or mot) for 7 days with warfarin. Ultrathin frozen sections were incubated with goat antiserum against rat osteocalcin at high dilutions (2 x 10(-4) to 2 x 10(-6). In calvaria and femur of untreated rats, endogenous osteocalcin was observed in osteoblasts (cytoplasm and nucleus) and in the collagenous matrix. Osteocalcin appeared progressively in osteoblasts and bone matrix in the mineralization front, then increased in the regions of extended calcification. Osteocalcin was also detected in osteocytes but was not as abundant as in osteoblasts. In bone samples of warfarin-treated rats, endogenous osteocalcin was only detected in bone matrix but not in osteoblasts. Furthermore, osteocalcin was only observed if antiserum was not very dilute (2 x 10(-2). In cartilage (hypertrophied and degenerative zones), osteocalcin was not observed in matrix and chondrocytes. However, it was found in the vicinity of matrix vesicles at the initial loci of calcification. Osteocalcin was never detected in the cartilage of warfarin-treated rats. Our results provide ultrastructural immunocytological evidence for the localization of endogenous osteocalcin in osteoblasts, the presence of osteocalcin in bone matrix and a direct gradient between the presence of osteocalcin and the calcification process. Osteocalcin is absent from cartilage, except possibly close to calcifying matrix vesicles. Warfarin inhibits the formation of osteocalcin.

Type X collagen gene expression in mouse chondrocytes immortalized by a temperature-sensitive simian virus 40 large tumor antigen.

“…Both hypertrophic chondrocytes and osteoblasts have the ability to induce mineralization of the extracellular matrix and to produce alka- line phosphatase (ALP) ~ (1), osteopontin (OP) (11,29,45), osteonectin (21), matrix Gla protein (MGP) (2,12,17), and the Ch21 protein (9). Bone Gla protein (BGP, also called osteocalcin), another osteoblast marker (35), is present at low levels in the calcified cartilage matrix surrounding hypertrophic chondrocytes in vivo (4,28,29). Studies with chondrocytes in culture have shown that after the cells became hypertrophic they produced type I collagen and BGP (3,8,24,37,41,42), which led to the suggestion that these cells could acquire osteoblastic properties.…”

Morphological and Immunocytochemical Characterization of Osteoblast Cultures from Long Bones of Neonatal Rats.

“…Although the presence of osteocalcin antigenicity in the nucleus appears very difficult to explain, we are inclined to believe that cultured cells obtained in our system were osteoblasts. The presence of ALP and osteocalcin in our cultured bone cells and the absence of any osteocalcin synthesis in fibroblasts, osteoclasts, and chondrocytes (BoIvIN et al, 1990) provided rationales for justification of this osteoblast phenotypic expression.…”

“…The existence of osteocalcin was demonstrated in the cultured rat osteosarcoma cell clone ROS 17/2, osteoblasts and bone matrix in the mineralization front (NISHIMOTO and PRICE, 1980;PRICE et al, 1981;BIANCO et al, 1985;GROOT et al, 1986;THAVARAJAH et al, 1986;CAMARDA et al, 1987;OHTA et al, 1989;BOIvIN et al, 1990). Osteocalcin was also detected in osteocytes.…”

“…BIANCO et al (1985) showed that endogenous osteocalcin was present in the cytoplasm and nucleus of osteoblasts and in the collagenous matrix. Moreover, CAMARDA et al (1987), OHTA et al (1989) and BoIvIN et al (1990) demonstrated that osteocalcin was present in the rough endoplamic reticulum and Golgi apparatus of osteocytes and osteoblasts by immunoelectron microscopy, and suggested that osteoblasts and osteocytes are the cells responsible for its synthesis. In our systems, osteocalcin was demonstrated in both the nucleus and cytoplasm of these cells.…”

“…Therefore, identification by morphological distinctions with phase microscopy, SEM and TEM, histochemical staining with ALP, and responsiveness to parathyroid hormone etc., have been employed to establish the exact nature of the cultured bone cells (WILLIAMS et al, 1980;WONG, 1980;NIJWEIDE et al, 1981;PARTRIDGE et al, 1983;AUF'MKOLK et al 1985;ROBEY and TERMINE, 1985;MARIE et al, 1989;SHAW et al, 1989;MASQUELIER et al, 1990). Moreover, osteocalcin was determined to be produced only by osteoblasts and osteocytes; therefore, immunocytochemical staining with a monoclonal or polyclonal antibody against osteocalcin provided a specific method of identification (BIANCO et al, 1985;GROOT et al, 1986;THAVARAJAH et al, 1986;CAMARDA et al, 1987;MARK et al, 1987;WANG et al, 1987WANG et al, , 1991VRIES et al, 1988;OHTA et al, 1989;BOIVIN et al, 1990).…”