Expression of Bone Morphogenetic Proteins (BMPs) in Fractured Mouse Bone Tissue: In Situ Hybridization with Polymerase Chain Reaction (PCR)-derived Antisense DNA Probe

摘要

We investigated the expression of bone morphogenetic protein (BMP)-2, -4 and -6 during fracture healing of mouse tibiae by in situ hybridization (ISH). Twelve-week-old male BALB/c mice were operated on to make a closed fracture on the proximal tibiae. On days 4, 7 and 14 after the operation, the fractured bones were excised, fixed with 4% PFA and decalcified with 20% EDTA to prepare 5-mum sections. For ISH we employed digoxigenin (DIG) labeled single-stranded DNA probes specific to BMP-2, -4 and -6 generated by uni-directional polymerase chain reaction (PCR) with antisense primer alone.On days 4-14 after fracture, BMP-2 signals were predominantly expressed in proliferating-hypertrophic chondrocytes and in osteoblasts on the surface of the marginal woven bone. Weak expression of BMP-4 and -6 was detected in spindle-shaped mesenchymal cells and at the site of chon-drocytic differentiation; it then decreased during the formation of woven bone. These data suggested that BMP-4 and -6 may be important in the early phase; BMP-2 contributed mainly in the mid to later phase of bone fracture repair. BMPs seemed to promote both proliferation and differentiation of mesenchymal cells through the paracrine/ autocrine mechanism.