Kazmers Nikolas H, McKenzie Jennifer A, Shen Tony S, Long Fanxin, Silva Matthew J
Department of Orthopaedic Surgery, Washington University, Campus Box 8233, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
Department of Orthopaedic Surgery, Washington University, Campus Box 8233, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
Bone. 2015 Dec;81:524-532. doi: 10.1016/j.bone.2015.09.002. Epub 2015 Sep 6.
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 3 days (Ptch1, Gli1 expression), and was markedly inhibited by GDC-0449 at 1 day and 3 days in the loaded and non-loaded ulnae. GDC-0449 did not affect Hh ligand expression (Shh, Ihh, Dhh) at 1 day, but decreased Shh expression by 37% at 3 days. GDC-0449 decreased woven bone volume (-37%) and mineral density (-17%) at 7 days. Dynamic histomorphometry revealed that the 7 day 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 3 days), osteoblastic differentiation (Osx expression at 1 day and 3 days), chondrogenic gene expression (Acan, Sox9, and Col2α1 expression at 1 day and 3 days), or bone resorption metrics (callus TRAP staining at 3 days, Rankl and Opg expression at 1 day and 3 days). To evaluate angiogenesis, vWF immunohistochemistry showed that GDC-0449 reduced fracture callus blood vessel density by 55% at 3 days, 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.
刺猬信号通路（Hh）在发育性骨生成中至关重要，最近的研究表明它在出生后调节成骨基因表达方面可能也发挥作用。然而，目前缺乏直接评估Hh抑制对出生后骨生成影响的研究。本研究利用周期性负荷诱导的尺骨应力性骨折模型，以评估Hh信号通路在应力性骨折愈合过程中对骨生成和血管生成有贡献这一假说。在负荷前，成年大鼠被给予GDC - 0449（维莫德吉——一种选择性Hh通路抑制剂；口服50mg/kg，每日两次）或赋形剂。在第3天，应激性骨折可使Hh信号通路上调（Ptch1、Gli1表达），在负荷和未负荷的尺骨中，GDC - 0449在第1天和第3天可显著抑制该通路。GDC - 0449在第1天不影响Hh配体表达（Shh、Ihh、Dhh），但在第3天可使Shh表达降低37%。GDC - 0449在第7天可使编织骨体积减少37%，矿物质密度降低17%。动态组织形态计量学显示，两组第7天的骨痂主要由编织骨组成。观察到的编织骨减少与Alpl和Ibsp表达降低同时发生，但与早期细胞增殖差异（通过第3天骨痂PCNA染色确定）、成骨细胞分化（第1天和第3天Osx表达）、软骨形成基因表达（第1天和第3天Acan、Sox9和Col2α1表达）或骨吸收指标（第3天骨痂TRAP染色、第1天和第3天Rankl和Opg表达）无关。为评估血管生成，vWF免疫组化显示，GDC - 0449在第3天可使骨折骨痂血管密度降低55%，这与Hif1α基因表达增加30%相关。动态组织形态计量分析表明，GDC - 0449也抑制板层骨形成。对负荷肢体（紧邻编织骨痂）的板层骨分析显示，GDC - 0449可显著降低矿物质沉积率（MAR）和骨形成率（BFR/BS）（分别降低17%和20%）。未负荷尺骨的板层BFR/BS也显著降低（37%）,表明Hh信号通路是正常骨塑形所必需的。总之，Hh信号通路在应力性骨折愈合的出生后骨生成中起重要作用，通过调节骨形成和血管生成直接介导其效应。
