IN-SITU HYBRIDIZATION TO SHOW SEQUENTIAL EXPRESSION OF OSTEOBLAST GENE MARKERS DURING BONE-FORMATION IN-VIVO

We investigated the sequence of expression of osteoblast gene markers during bone formation in vivo by in situ hybridization. Cylindrical le sions were induced in the femora of sheep with titanium analytic bone implants that allow removal of serial core samples to study bone forma tion. At 2 weeks (2W), granulation tissue made up of spindle-shaped ce lls had partially replaced the blood clot. Islands of osseous tissue, first noted in the periphery of the ingrowing tissue at 3W, became the predominant tissue by 6W, The surfaces of newly forming bone at 3W we re apposed by cuboidal cells, which in some areas were several layers thick. By 6W, most of the cells lining bone trabeculae had assumed a f lattened morphology. The temporal and spatial distribution of osteobla st gene markers was examined by in situ hybridization with nonradioact ive digoxigenin probes for alpha(1)(I) procollagen, alkaline phosphata se (ALP), osteopontin (OP), and bone Gla protein (BGP). The spindle-sh aped cells in the granulation tissue expressed mRNA for alpha(1)(I) pr ocollagen, ALP, and OP but not BGP, suggesting that they may be osteob last precursor cells. alpha(1)(I) procollagen mRNA was strongly expres sed by all cells on the surface of bone, with a peak intensity at 3W a nd then reducing sharply by 6W. Initially, only pockets of cuboidal ce lls on bone surfaces expressed ALP mRNA, with a peak intensity at 5W. Similarly, only a proportion of cuboidal cells expressed OP mRNA early in bone formation, but the number of cells expressing OP mRNA increas ed with time. Clumps of cuboidal cells expressed BGP mRNA only when bo ne was present, and the degree of expression increased with the amount of bone formed. This model allows the study of temporal and spatial s equence of gene expression in cells participating in osteogenesis. The temporal sequence is similar to that shown in vitro in other models o f mineralization. The geographic localization of cells expressing mRNA for alpha(1)(I) procollagen, ALP, OF, and BGP implies subspecializati on of osteoblasts in bone formation.