Changes in ephrin gene expression during bone healing identify a restricted repertoire of ephrins mediating fracture repair.

To identify the repertoire of ephrin genes that might regulate endochondral bone fracture repair, we examined changes in ephrin ligand and receptor (Eph) gene expression in fracture callus tissues during bone fracture healing. Ephrin and Eph proteins were then localized in the fracture callus tissues present when changes in gene expression were observed. Ephrin gene expression was widespread in fracture tissues, but the repertoire of ephrin genes with significant changes in expression that might suggest a regulatory role in fracture callus development was restricted to the ephrin A family members Epha4, Epha5 and the ephrin B family member Efnb1. After 3 weeks of healing, Epha4 fracture expression was downregulated from 1.3- to 0.8-fold and Epha5 fracture expression was upregulated from 1.2- to 1.5-fold of intact contralateral femur expression, respectively. Efnb1 expression was downregulated from 1.5- to 1.2-fold after 2 weeks post-fracture. These ephrin proteins were localized to fracture callus prehypertrophic chondrocytes and osteoblasts, as well as to the periosteum and fibrous tissues. The observed positive correlation between mRNA levels of EfnB1 with Col10 and Epha5 with Bglap, together with colocalized expression with their respective proteins, suggest that EfnB1 is a positive mediator of prehypertrophic chondrocyte development and that Epha5 contributes to osteoblast-mediated mineralization of fracture callus. In contrast, mRNA levels of Epha4 and Efnb1 correlated negatively with Bglap, thus suggesting a negative role for these two ephrin family members in mature osteoblast functions. Given the number of family members and widespread expression of the ephrins, a characterization of changes in ephrin gene expression provides a basis for identifying ephrin family members that might regulate the molecular pathways of bone fracture repair. This approach suggests that a highly restricted repertoire of ephrins, EfnB1 and EphA5, are the major mediators of fracture callus cartilage hypertrophy and ossification, respectively, and proposes candidates for additional functional study and eventual therapeutic application.