Hedgehog signaling mediates woven bone formation and vascularization during stress fracture healing

Abstract

Hedgehog (Hh) signaling is critical in developmental osteogenesis, and recent studies suggest it may also play a role in regulating osteogenic gene expression in the post-natal setting. However, there is a void of studies directly assessing the effect of Hh inhibition on post-natal osteogenesis. This study utilized a cyclic loading-induced ulnar stress fracture model to evaluate the hypothesis that Hh signaling contributes to osteogenesis and angiogenesis during stress fracture healing. Immediately prior to loading, adult rats were given GDC-0449 (Vismodegib — a selective Hh pathway inhibitor; 50mg/kg orally twice daily), or vehicle. Hh signaling was upregulated in response to stress fracture at 3days (Ptch1, Gli1 expression), and was markedly inhibited by GDC-0449 at 1day and 3days in the loaded and non-loaded ulnae. GDC-0449 did not affect Hh ligand expression (Shh, Ihh, Dhh) at 1day, but decreased Shh expression by 37% at 3days. GDC-0449 decreased woven bone volume (−37%) and mineral density (−17%) at 7days. Dynamic histomorphometry revealed that the 7day callus was composed predominantly of woven bone in both groups. The observed reduction in woven bone occurred concomitantly with decreased expression of Alpl and Ibsp, but was not associated with differences in early cellular proliferation (as determined by callus PCNA staining at 3days), osteoblastic differentiation (Osx expression at 1day and 3days), chondrogenic gene expression (Acan, Sox9, and Col2α1 expression at 1day and 3days), or bone resorption metrics (callus TRAP staining at 3days, Rankl and Opg expression at 1day and 3days). To evaluate angiogenesis, vWF immunohistochemistry showed that GDC-0449 reduced fracture callus blood vessel density by 55% at 3days, which was associated with increased Hif1α gene expression (+30%). Dynamic histomorphometric analysis demonstrated that GDC-0449 also inhibited lamellar bone formation. Lamellar bone analysis of the loaded limb (directly adjacent to the woven bone callus) showed that GDC-0449 significantly decreased mineral apposition rate (MAR) and bone formation rate (BFR/BS) (−17% and −20%, respectively). Lamellar BFR/BS in the non-loaded ulna was also significantly decreased (−37%), indicating that Hh signaling was required for normal bone modeling. In conclusion, Hh signaling plays an important role in post-natal osteogenesis in the setting of stress fracture healing, mediating its effects directly through regulation of bone formation and angiogenesis.