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 3d (Ptch1, Gli1 expression), and was markedly inhibited by GDC-0449 at 1d and 3d in the loaded and non-loaded ulnae. GDC-0449 did not affect Hh ligand expression (Shh, Ihh, Dhh) at 1d, but decreased Shh expression by 37% at 3d. GDC-0449 decreased woven bone volume (−37%) and mineral density (−17%) at 7d. Dynamic histomorphometry revealed that the 7d 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 3d), osteoblastic differentiation (Osx expression at 1d and 3d), chondrogenic gene expression (Acan, Sox9, and Col2α1 expression at 1d and 3d), or bone resorption metrics (callus TRAP staining at 3d, Rankl and Opg expression at 1d and 3d). To evaluate angiogenesis, vWF immunohistochemistry showed that GDC-0449 reduced fracture callus blood vessel density by 55% at 3d, 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.

Exogenous hedgehog antagonist delays but does not prevent fracture healing in young mice

“…This result is consistent with a previous study, wherein we noted that GDC treatment significantly reduced expression of osteogenic genes and periosteal bone formation in normal rat ulnae (i.e., not subjected to mechanical injury) [28]. These findings are also consistent with the findings of Ohba et al [36], who reported that treatment of mice with a Hh inhibitor (cyclopamine, a Smo antagonist) decreased bone formation and bone mass.…”

“…On the other hand, deletion in the osteoblast lineage (using Col1Cre) significantly reduced the callus bone volume at day 28, indicating arole for osteoblastic Hh signaling in bone formation during fracture healing. Consistent with this latter result, we previously found that the Smo antagonist GDC-0449 diminished intramembranous bone formation after stress fracture [28]. Taken together, the previous studies indicate the Hh may play a role in osteogenesis during bone healing.…”

“…The treatment group received GDC-0449 (ChemieTek, Indianapolis, IN; 50mg/kg; 20mg/mL dissolved in DMSO), hereafter referred to as GDC, twice daily by oral gavage (#20 gavage needle) until designated euthanasia time points. This regimen was selected based on our previous work [28] and pilot studies demonstrating suppression of Ptch1 and Gli1 expression in bone (confirmed in Results below). The control group received equivalent volume (2.5 ml/kg) of DMSO vehicle, hereafter referred to as Veh, twice daily by oral gavage.…”

“…First, genetic inactivation of Hh in osteoblasts blunted bone matrix deposition during endochondral healing in mice [27]. Second, the use of the pharmacological Smo antagonist, GDC-0449, reduced osteogenesis and angiogenesis during stress fracture healing in rats [28]. Our goal here was to extend these previous studies and assess the functional importance of Hh signaling in endochondral fracture healing.…”

Network-Based Method for Identifying Co-Regeneration Genes in Bone, Dentin, Nerve and Vessel Tissues

“…As for its specific role for bone and vessel co-regeneration, expressed by MSCs, IHH has been confirmed to be overexpressed during the repair and regeneration of bone, indicating its specific role of bone regeneration [64]. At the same time, during the regeneration of bone, together with GLI1 as we have analyzed above, our predicted gene IHH has also been confirmed to contribute to vascularization mediating by hedgehog signaling pathway [65]. As we have mentioned above, MET (ENSP00000317272) has been confirmed to participate in the co-regeneration of bone and nerve [51,52].…”