Modulation of osteonectin and osteopontin expression during fracture healing of mouse bone tissue

We investigated the expression of osteonectin and osteopontin during fracture healing of mouse tibiae by in situ hybridization (ISH) and the effects of fibrin(ogen) on the expression of bone matrix proteins by Northern blot analysis. Twelve-week-old male BALB/c mice were operated on to make a closed fracture on the proximal tibiae. On days 2, 4, 7 and 14 after the operation, the fractured bones were excised, fixed with 4% paraformaldehyde (PFA) and decalcified with 20% EDTA to prepare 5-mu m sections. Digoxigenin (DIG) labeled single-stranded DNA probes generated by uni-directional polymerase chain reaction (PCR) were used for ISH. Immunohistochemistry revealed fibrin(ogen) at the site of fracture hematoma 2-4 days after fracture and subsequent accumulation of mesenchymal cells. On days 4-14 after the fracture, osteonectin signals were predominantly expressed in proliferating chondrocytes at the endochondral ossification site and in osteoblasts of the marginal woven bone. Osteopontin was expressed on osteoblasts lining the surface of the marginal woven bone at the mid-late phase of fracture healing. In vitro, Northern blot analysis showed that treatment of the mesenchymal cells, C3H10T1/2, with fibrin(ogen) enhanced the steady-state level of osteonectin and osteopontin gene expression. These data suggested that fibrin(ogen) in the hematoma may be important for inducing bone matrix genes in immature mesenchymal cells at the early phase of fracture repair.