Interleukin-6 (IL-6) deficiency enhances intramembranous osteogenesis following stress fracture in mice

Interleukin-6 (IL-6) is highly upregulated in response to skeletal injury, suggesting it plays a role in the inflammatory phase of fracture repair. However, the impact of IL-6 on successful repair remains incompletely defined. Therefore, we investigated IL-6 in fracture repair using 12-week old IL-6 global knockout mice (IL-6 KO) and two models of fracture repair: full fracture and stress fracture. Callus formation 14 days after full fracture did not differ between IL-6 knockout mice and controls. However, IL-6 KO mice had an enhanced response 7 days after stress fracture compared to control, with increased callus (p=0.020) and bone formation (p=0.045). IL-6 KO did not alter the recruitment of neutrophils or macrophages to the stress fracture callus. IL-6 KO also did not alter the number of osteoclasts in the stress fracture callus. Based on RNA-seq, IL-6 KO resulted in only modest alterations to the gene expression at early time points after stress fracture. Wnt1 was more highly upregulated in IL-6 KO callus at both day 1 (fold change 12.5 vs. 5.7) and day 3 (fold change 4.7 vs. 1.9) compared to controls. Finally, using tibial compression to induce bone formation, we found that IL-6 KO directly impacted osteoblast function, increasing the propensity for woven bone formation. Herein, we report that IL-6 knockout enhanced formation of callus and bone following stress fracture injury, likely through direct action on the osteoblast's ability to produce woven bone. This suggests a novel role of IL-6 as a suppressor of intramembranous bone formation. Overall design: 36 samples, 12 non-loaded/intact control ulnae from IL6 WT (n=6) and IL6 KO (n=6). 12 samples from a stress fracture ulna at day 1 (24h) after injury from IL6 WT (n=6) and IL6 KO (n=6). 12 samples from stress fracture ulnae at day 3 (72h) after injury from IL6 WT (n=6 and IL6 KO (n=6) mice. All groups were a mix of male and female mice.

白细胞介素-6（Interleukin-6, IL-6）在骨骼损伤时呈现显著上调，提示其参与骨折修复的炎症阶段。然而，白细胞介素-6对骨折修复成功结局的影响仍未完全阐明。
为此，本研究以12周龄的白细胞介素-6全身敲除小鼠（IL-6 KO）为研究对象，采用完全性骨折与应力性骨折两种骨折修复模型，探究白细胞介素-6在骨折修复中的作用。
完全性骨折造模后14天，白细胞介素-6敲除小鼠与对照组的骨痂形成量无显著差异。但在应力性骨折造模后7天，白细胞介素-6敲除小鼠的骨修复反应较对照组增强，骨痂面积（p=0.020）与骨形成量（p=0.045）均有所升高。
白细胞介素-6敲除并未改变中性粒细胞与巨噬细胞向应力性骨折骨痂的募集情况。
白细胞介素-6敲除也未对应力性骨折骨痂中的破骨细胞数量产生影响。
通过RNA测序（RNA-seq）分析可见，白细胞介素-6敲除仅在应力性骨折造模后的早期阶段对基因表达产生轻微调控作用。
与对照组相比，白细胞介素-6敲除小鼠骨痂中的Wnt1在造模后第1天（倍数变化12.5 vs 5.7）与第3天（倍数变化4.7 vs 1.9）均呈现更高水平的上调。
最后，通过胫骨加压诱导骨形成的实验，本研究发现白细胞介素-6敲除可直接影响成骨细胞功能，提升编织骨形成的倾向。
本研究结果表明，白细胞介素-6敲除可增强应力性骨折损伤后的骨痂与骨形成，这一效应可能通过直接调控成骨细胞产生编织骨的能力实现。
这提示白细胞介素-6作为膜内骨形成的抑制因子，发挥了此前未被报道的全新功能。
实验整体设计：共纳入36份样本。其中，6只白细胞介素-6野生型（IL6 WT）小鼠与6只白细胞介素-6敲除（IL6 KO）小鼠的未加载完整尺骨作为对照，共12份样本；造模后第1天（24小时）的应力性骨折尺骨样本共12份，两组小鼠各6只；造模后第3天（72小时）的应力性骨折尺骨样本共12份，两组小鼠各6只。所有组别均混合了雄性与雌性小鼠。