U sing in situ hybridisation and the terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end-labelling (TUNEL) reaction in rats with osteonecrosis of the femoral head we have studied the effect of ischaemia on the gene expression of the stress proteins oxygen-regulated protein 150 (ORP150) and haemoxygenase 1 (HO1) and the death mechanism of the cells involved in osteonecrosis. Both ORP150 and HO1 have been reported to have important roles in the successful adaptation to oxygen deprivation.ORP150 and HO1 mRNA expression was induced by ischaemia in osteoblasts and osteocytes. In proliferative chondrocytes, these signals were detected constitutively. During the development of ischaemic osteonecrosis, the mechanism of cell death was apoptosis as indicated by DNA fragmentation and the presence of apoptotic bodies in osteocytes, chondrocytes and bone-marrow cells. After the initial ischaemic event, expression of ORP150 and HO1 mRNA, the TUNEL-positive reaction and empty lacunae were found sequentially. These findings were exclusive and may be considered to be markers for each stage in the development of osteonecrosis. Traumatic and non-traumatic osteonecrosis of the femoral head is assumed to have an ischaemic origin although the pathomechanism of non-traumatic osteonecrosis is still unclear. Even with variable aetiological factors, the pathology is quite similar.1 After the initial ischaemic events, death of bone and marrow elements occurs while the articular cartilage is intact. Although osteonecrosis is often diagnosed histologically under light microscopy by the presence of empty lacunae, the precise cellular and molecular basis of these changes in bone has not been clarified and elucidation of this may help in the understanding of the pathomechanism of non-traumatic osteonecrosis. It has been reported that deprivation of environmental oxygen induces expression of a set of stress proteins 2,3 which are believed to have an important role in protecting cellular biosynthetic activities under stressful circumstances. Recently, a 150-kD polypeptide termed oxygen-regulated protein (ORP150) has been isolated and cloned and was found to be induced selectively in astrocytes by hypoxia. 3 In this respect ORP150 differs from haemoxygenase 1 (HO1) which shows enhanced expression in response to hypoxia, as well as other stimuli such as heat shock, heavy metals, endotoxin and inflammatory cytokines. 4,5 ORP150 plays a pivotal role in the cytoprotective cellular mechanisms triggered by oxygen deprivation, one of which is the inhibition of apoptosis. 6We have used in situ hybridisation and the terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end-labelling (TUNEL) reaction, to demonstrate the spatial localisation and temporal expression of mRNAs for ORP150 and HO1, and TUNEL-positive cells during the development of ischaemic osteonecrosis. Materials and MethodsThe experimental procedures in this study were undertaken in compliance with the USA guidelines for the care and use of animals. We used 42 male ...

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