Immunocytochemical localization of γ-carboxyglutamic acid-containing proteins (osteocalcin) in rat bone and dentin

Abstract

Gla-protein or osteocalcin is one of the most abundant noncollagenous matrix proteins found in bone and dentin. The present study describes, with high resolution, the intracellular and extracellular distribution of Gla-protein in alveolar bone and incisor dentin. Sections of tissues embedded in Lowicryl K4M were incubated with rabbit antibodies to rat dentin Gla-protein. The site of the specific antigen-antibody reaction was revealed by the protein A-gold complex. Labeling was detected over bone and dentin while fewer gold particles were present over prebone and predentin. Gold particles were also seen over the protein synthetic organelles (rough endoplasmic reticulum, Golgi apparatus) of osteoblasts and odontoblasts. These findings confirm, with improved resolution, previous light immunohistochemical studies, and offer the possibility to examine the secretory pathway of the protein.

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

“…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).…”

Lectin Binding in Bone Matrix of Adult Rats with Special Reference to Cement Lines.

“…Although several immunohistochemical studies on the non-collagenous molecules such as bone sialoprotein (Bianco et al 1991), osteopontin (Mark et al 1987), osteocalcin (Camarda et al 1987), dermatan sulphate proteoglycan (Poole et al 1986), tenascin (Thesleff et al 1988), osteonectin (Bianco et al 1985), a2-HS glycoprotein (Dickson et al 1975) have been reported, molecules specific for the cement line have not been found yet.…”

Immunocytochemistry of matrix proteins in calcified tissues: functional biochemistry on section

“…Initial immunogold studies on calcified tissues dealt with the distribution of enamel proteins in teeth ( Figure 7) (Nanci et al 1984a;1984b; and osteocalcin in bone and dentin (Bianco et al 1985;Camarda et al 1987;Gorter de Vries et al 1987). It has since been used to characterize the presence and distribution of a number of matrix molecules in normal and pathological calcified tissues of various animal species, ranging from invertebrates to mammals.…”