THE ROLE OF IRON IN THE INFLAMMATORY STAGE OF HETEROTOPIC OSSIFICATION

Heterotopic ossification (HO) is bone formation at abnormal anatomical sites, developing in three main stages: inflammation, chondrogenesis and osteogenesis. As iron homeostasis plays an important role in inflammatory diseases and bone formation, we aimed to investigate the impact of iron levels on HO development.

For this purpose, wild type mice were fed with either low (<10 ppm Fe), normal (200 ppm Fe) or high (25,000 ppm Fe) iron diet for 6 weeks starting from weaning on. Afterwards, HO was induced by injection of recombinant BMP-2 into the M. tibialis anterior. HO sites were analyzed by flow cytometry, proteome cytokine profiler (day 3) and µCT analysis (day 14).

In mice fed with high iron diet, iron accumulated in the inner organs such as liver (2001.6 vs 280.7 µg Fe/g dry tissue, p<0.001) as well as in the muscle (0.14 vs 0.69 µg Fe/ mg protein, p<0.001). High iron diet led to a 2.6-fold increase in HO 14 days after BMP-2 injection (p<0.05), while low iron diet led to a 65 %-decrease of bone formation (p<0.05) compared to normal iron diet.

Analyzing the inflammatory phase of HO, mice under high iron diet showed enhanced expression of pro-inflammatory cytokines (e.g. IFNγ: 2-fold increase; p<0.05, IL-6: 1.7-fold; p<0.05) and more infiltrated CD11b+ myeloid cells and classical macrophages (p<0.05). Neutrophils and non-classical macrophages were decreased under high-iron diet (p<0.05).

Iron levels affect the development of HO, most likely already in the early inflammatory stage of HO by increasing pro-inflammatory cytokines and immune cells.