CELLULAR EXPRESSION OF BONE-RELATED PROTEINS DURING IN-VITRO OSTEOGENESIS IN RAT BONE-MARROW STROMAL CELL-CULTURES

Rat bone marrow stromal cells comprise a heterogeneous mixture of cell lineages including osteoblastic cells. When grown in the presence of ascorbic acid, beta-glycerophosphate and 10(-8)M dexamethasone, osteop rogenitor cells within the population divide and differentiate to form bone nodules (Maniatopoulos et al., 1988, Cell Tissue Res., 254:317-3 30; Aubin et al., 1990, J. Bone Miner. Res., 5:S81) providing a useful model to investigate temporal and spatial changes in expression of os teoblastic markers. Immunocytochemistry was combined with Northern blo tting, enzymatic assay, and radioimmunoassay to analyze the expression of bone-related proteins during the growth and differentiation sequen ce. By mRNA levels, protein production and/or enzymatic activity, expr ession of osteocalcin, bone sialoprotein, and alkaline phosphatase inc reased concomitantly with the development of bone nodules, while osteo pontin mRNA levels decreased and those of SPARC/osteonectin did not ch ange significantly. In older cultures with mineralizing nodules, mRNA levels for alkaline phosphatase and bone sialoprotein, but not osteoca lcin, declined. Immunolabeling revealed that cells in early cultures s tained poorly for SPARC/osteonectin and strongly for thrombospondin. L ater, SPARC/ osteonectin staining increased in most cells, while throm bospondin staining could be seen in both matrix and in cells, but with marked intercellular variability in intensity. At all time points stu died, osteoblasts within bone nodules stained homogeneously for thromb ospondin and alkaline phosphatase, and with marked heterogeneity of in tensity amongst cells for SPARC/osteonectin and osteocalcin. Labelling with RCC455.4, a monoclonal antibody raised against rat calvaria cell s which intensely labels osteoblasts and osteocytes (Turksen et al., 1 992, J. Histochem. Cytochem., 40:1339-1352), co-localized with osteoca lcin. Alkaline phosphatase activity and the amount of osteocalcin dete rmined by both radioimmunoassay and immunolabelling decreased in very late cultures, a time corresponding to appearance of fully mineralized nodules. These studies indicate that the bone marrow stromal cell sys tem is a useful model to study the temporal and spatial expression or bone-related proteins during osteogenesis and formation, mineralizatio n, and maturation of bone nodules. Further, immunolabelling at the ind ividual cell and single bone nodule level allowed discrimination of ma rked variability of expression of osteoblast markers during the differ entiation sequence. (C) 1994 Wiley-Liss, inc.