Immunohistochemical investigations on the differentiation marker protein E11 in rat calvaria, calvaria cell culture and the osteoblastic cell line ROS 17/2.8

Until now, many extracellular matrix proteins, e.g. osteopontin and osteone ctin, have been used to determine a cell's osteogenic maturation. The disad vantage in evaluation of these proteins is their relative wide-ranging appe arance throughout the osteogenic differentiation process. Thus, the aim of this study was to establish an immunohistochemical setup using E11, a marke r that binds selectively to cells of the late osteogenic cell lineage. In a ddition, the histochemical expression of the bone matrix proteins osteonect in, osteopontin and fibronectin was compared to that of E11 using monoclona l antibodies. For light microscopical detection of osteogenic markers in cu ltured cells we developed a simple paraffin technique using a fibrin glue a s embedding medium. This allows the handling of cultured cells such as a ti ssue sample and includes the use of stored biological specimens for further immunohistochemical experiments. We used newborn rat calvariae for whole t issue preparations and for isolation and cultivation of bone cells. In addi tion, we included the rat osteosarcoma cell line ROS 17/2.8 in this study. For the first time, we have localised E11 in osteocytes of rat calvaria pre parations at the electron microscopical level. E11 was detected at plasma m embranes of osteocytes and their processes, but not at those Of osteoblasts . Accompanying experiments with cultured newborn rat calvaria cells and ROS 17/2.8 cells revealed E11 reactivity on a subset of cells. The results obt ained confirm the suitability of the differentiation marker E11 as a sensit ive instrument for the characterisation of bone cell culture systems.