BONE-MATRIX PROTEINS IN OSTEOGENESIS AND REMODELING IN THE NEONATAL RAT MANDIBLE AS STUDIED BY IMMUNOLOCALIZATION OF OSTEOPONTIN, BONE SIALOPROTEIN, ALPHA-2HS-GLYCOPROTEIN AND ALKALINE-PHOSPHATASE

The neonatal rat mandible was used as a model to study bone formation, mineralization, quiescence, and resorption, using immunolocalization and a variety of tissue-processing techniques. Monospecific antibodies for osteopontin (OPN), bone sialoprotein (BSP), alkaline phosphatase (AP) and alpha(2)HS-glycoprotein (alpha(2)HS-GP) were used on fixed pa raffin-embedded tissue, fixed frozen tissue and unfixed frozen tissue, Immunostaining was correlated with mineral content by two procedures, the von Kossa and the morin techniques. Morin fluorescence was used w ith secondary immunostaining to provide a way of closely correlating b one matrix proteins and matrix mineralization. Co-immunolocalization p rocedures were used to compare the sites of bone proteins in the matri x. AP was found earliest during osteogenic cell differentiation, appea ring in the preosteoblasts, followed by OPN and BSP, which first appea red in osteoblasts. alpha(2)HS-GP expression was not observed in cells . The results provide clear evidence for the presence of OPN in osteoi d, while BSP and alpha(2)HS-GP were confined to the mineralized matrix . Immunostaining of bone proteins is highly technique-dependent: immun olocalization investigations required several methods of approach to e nsure adequate demonstration of these proteins in cells and matrix. Th e results support the contention that osteopontin is multifunctional i n bone metabolism, and that alpha(2)HS-GP, though produced in the live r, is abundant in bone matrix and may also have a function in bone met abolism.