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In situ hybridization to show sequential expression of osteoblast gene markers during bone formation in vivo
Author(s) -
Zhou H.,
Choong P.,
McCarthy R.,
Chou S.T.,
Martin T.J.,
Ng Kong Wah
Publication year - 1994
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1002/jbmr.5650090922
Subject(s) - in situ hybridization , osteoblast , osteopontin , alkaline phosphatase , bone cell , biology , procollagen peptidase , granulation tissue , columnar cell , microbiology and biotechnology , cuboidal cell , gene expression , chemistry , anatomy , pathology , epithelium , endocrinology , biochemistry , in vitro , gene , immunology , wound healing , medicine , genetics , enzyme
We investigated the sequence of expression of osteoblast gene markers during bone formation in vivo by in situ hybridization. Cylindrical lesions were induced in the femora of sheep with titanium analytic bone implants that allow removal of serial core samples to study bone formation. At 2 weeks (2W), granulation tissue made up of spindle‐shaped cells 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 were apposed by cuboidal cells, which in some areas were several layers thick. By 6W, most of the cells lining bone trabeculae had assumed a flattened morphology. The temporal and spatial distribution of osteoblast gene markers was examined by in situ hybridization with nonradioactive digoxigenin probes for α 1 (I) procollagen, alkaline phosphatase (ALP), osteopontin (OP), and bone Gla protein (BGP). The spindle‐shaped cells in the granulation tissue expressed mRNA for α 1 (I) procollagen, ALP, and OP but not BGP, suggesting that they may be osteoblast precursor cells. α 1 (I) procollagen mRNA was strongly expressed by all cells on the surface of bone, with a peak intensity at 3W and then reducing sharply by 6W. Initially, only pockets of cuboidal cells 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 increased with time. Clumps of cuboidal cells expressed BGP mRNA only when bone was present, and the degree of expression increased with the amount of bone formed. This model allows the study of temporal and spatial sequence of gene expression in cells participating in osteogenesis. The temporal sequence is similar to that shown in vitro in other models of mineralization. The geographic localization of cells expressing mRNA for α 1 (I) procollagen, ALP, OP, and BGP implies subspecialization of osteoblasts in bone formation.